125p5c8: tissue specific protein highly expressed in various cancers

ABSTRACT

A novel gene (designated 125P5C8) and its encoded protein are described. While 125P5C8 exhibits tissue specific expression in normal adult tissue, it is aberrantly expressed multiple cancers including prostate, bladder, kidney and colon cancers. Consequently, 125P5C8 provides a diagnostic and/or therapeutic target for cancers, and the 125P5C8 gene or fragment thereof, or its encoded protein or a fragment thereof used to elicit an immune response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.09/809,638 filed Mar. 14, 2001, which is now U.S. Pat. No. 7,271,240.The contents of this document are incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The invention described herein relates to a novel gene and its encodedprotein, termed 125P5C8, and to diagnostic and therapeutic methods andcompositions useful in the management of various cancers that express125P5C8.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of theseFigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer ResSep. 2, 1996 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad SciUSA. Dec. 7, 1999; 96(25): 14523-8) and prostate stem cell antigen(PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

SUMMARY OF THE INVENTION

The present invention relates to a novel gene, designated 125P5C8, thatis over-expressed in multiple cancers listed in Table I. Northern blotexpression analysis of 125P5C8 gene expression in normal tissues shows arestricted expression pattern in adult tissues. The nucleotide (FIG. 2)and amino acid (FIG. 2 and FIG. 3) sequences of 125P5C8 are provided.The tissue-related profile of 125P5C8 in normal adult tissues, combinedwith the over-expression observed in prostate and other tumors, showsthat 125P5C8 is aberrantly over-expressed in at least some cancers, andthus serves as a useful diagnostic and/or therapeutic target for cancersof the tissues such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 125P5C8 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding125P5C8-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80,85, 90, 95, 100 or more than 100 contiguous amino acids of a125P5C8-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the125P5C8 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the 125P5C8 genes, mRNAs, or to125P5C8-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 125P5C8. Recombinant DNA moleculescontaining 125P5C8 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 125P5C8gene products are also provided. The invention further providesantibodies that bind to 125P5C8 proteins and polypeptide fragmentsthereof, including polyclonal and monoclonal antibodies, murine andother mammalian antibodies, chimeric antibodies, humanized and fullyhuman antibodies, and antibodies labeled with a detectable marker.

The invention further provides methods for detecting the presence andstatus of 125P5C8 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 125P5C8.A typical embodiment of this invention provides methods for monitoring125P5C8 gene products in a tissue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 125P5C8such as prostate cancers, including therapies aimed at inhibiting thetranscription, translation, processing or function of 125P5C8 as well ascancer vaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. 125P5C8 SSH sequence. The SSH experiment was performed with cDNAdigested with DPN II. The 125P5C8 sequence contains 287 bp.

FIG. 2A-B. The cDNA and amino acid sequence of 125P5C8.

FIG. 3. The amino acid sequence encoded by the open reading frame of thenucleic acid sequence set forth in FIG. 2A-B.

FIG. 4A-B. Alignment of 125P5C8 with AK025164 (FIG. 4A) and the yeastprotein YCR017 (FIG. 4B) using the BLAST function (NCBI).

FIG. 5. High expression of 125P5C8 in prostate tissues. RT-PCR analysisshows high expression in the prostate cancer xenografts, normalprostate, prostate cancer, and to a lower extent in kidney, colon andbladder cancer specimens. Results are shown for 26 cycles (upper panel)and 30 cycles (lower panel). Lanes represent: VP1: Liver, kidney, lung;VP2: Stomach, spleen, pancreas; XeP: Xenograft pool: LAPC4AD, LAPC4AI,LAPC9AD, LAPC9AI; N.Pr.: Normal prostate pool; PrCa: Prostate cancerpool; BlCa: Bladder cancer pool; KiCa: Kidney cancer pool; CoCa: Coloncancer pool; LuCa: Lung cancer patient.

FIG. 6A-C. Expression of 125P5C8 in normal tissues (FIGS. 6A-B) and inprostate cancer xenografts (FIG. 6C). Two multiple tissue northern blots(Clontech) and a LAPC xenograft blot with 2 μg of mRNA/lane were probedwith the 125P5C8 SSH fragment. Size standards in kilobases (kb) areindicated on the side. The results show high expression of a 3 kbtranscript in normal prostate and prostate cancer xenografts LAPC4AI andLAPC9AI. Lower expression was detected in normal kidney and colon. Lanesrepresent: (FIG. 6A) 1. Heart; 2. Brain; 3. Placenta; 4. Lung; 5. Liver;6. Skeletal Muscle; 7. Kidney; 8. Pancreas; (FIG. 6B) 1. Spleen; 2.Thymus; 3. Prostate; 4. Testis; 5. Ovary; 6. Small Intestine; 7. Colon;8. Leukocytes; (FIG. 6C) 1. Normal Prostate; 2. LAPC-4 AD; 3. LAPC-4 AI;4. LAPC-9 AD; 5. LAPC-9 AI.

FIG. 7. Expression of 125P5C8 in prostate cancer patient tissues. RNAwas extracted from normal prostate, normal adjacent to tumor, and tumortissues. Northern blots with 10 μg of total RNA were probed with the125P5C8 SSH fragment. Size standards in kilobases are on the side.Results show expression in tumor-normal pairs with overexpression inpatient 1 tumor. Pt 1=Patient 1—Gleason 7; Pt 2=Patient 2—Gleason 7; Pt3=Patient 3—Gleason 7; Pt 4=Patient 4—Gleason 8; Pt 5=Patient 5—Gleason7; NP=Normal prostate; N=Normal adjacent tissue; T=Tumor.

FIG. 8. Expression of 125P5C8 in kidney cancer patient tissues. RNA wasextracted from normal kidney, normal adjacent to tumor, and tumortissues. Northern blots with 10 μg of total RNA were probed with the125P5C8 SSH fragment. Size standards in kilobases are on the side.Results show down-regulation of 125P5C8 in kidney tumor tissues. Lanesrepresent: Patient 1—papillary cell, grade 1; Patient 2—papillaryadenocarcinoma, nuclear, grade 3; Patient 3—clear cell, Fuhrman grade 2of 4; Patient 4—clear cell, grade III; Patient 5—clear cell, gradeII/IV; Patient 6—clear cell, grade 3; Patient 7—clear cell, gradeIII/IV; Patient 8—chromophobe cell type, grade IV ; Patient 9—metastasisto chest wall. NK=Normal kidney; N=Normal adjacent tissue; T=Tumor.

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections

-   I.) Definitions-   II.) Properties of 125P5C8.-   III.) 125P5C8 Polynucleotides

III.A.) Uses of 125P5C8 Polynucleotides

III.A.1.) Monitoring of Genetic Abnormalities

III.A.2.) Antisense Embodiments

III.A.3.) Primers and Primer Pairs

III.A.4.) Isolation of 125P5C8-Encoding Nucleic Acid Molecules

III.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

-   IV.) 125P5C8-related Proteins

IV.A.) Motif-bearing Protein Embodiments

IV.B.) Expression of 125P5C8-related Proteins

IV.C.) Modifications of 125P5C8-related Proteins

IV.D.) Uses of 125P5C8-related Proteins

-   V.) 125P5C8 Antibodies-   VI.) 125P5C8 Transgenic Animals-   VII.) Methods for the Detection of 125P5C8-   VIII.) Methods for Monitoring the Status of 125P5C8-related Genes    and Their Products-   IX.) Identification of Molecules That Interact With 125P5C8-   X.) Therapeutic Methods and Compositions

X.A.) 125P5C8 as a Target for Antibody-Based Therapy

X.B.) Anti-Cancer Vaccines

-   XI.) Inhibition of 125P5C8 Protein Function

XI.A.) Inhibition of 125P5C8 With Intracellular Antibodies

XII.B.) Inhibition of 125P5C8 with Recombinant Proteins

XI.C.) Inhibition of 125P5C8 Transcription or Translation

XI.D.) General Considerations for Therapeutic Strategies

-   XII.) KITS

I.) Definitions:

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers that have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1-C2 disease under the Whitmore-Jewettsystem, and stage T3-T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 125P5C8 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 125P5C8. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a125P5C8-related protein). For example an analog of the 125P5C8 proteincan be specifically bound by an antibody or T cell that specificallybinds to 125P5C8.

The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-125P5C8antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

As used herein, an “antibody fragment” is defined as at least a portionof the variable region of the immunoglobulin molecule that binds to itstarget, i.e., the antigen-binding region. In one embodiment itspecifically covers single anti-125P5C8 antibodies and clones thereof(including agonist, antagonist and neutralizing antibodies) andanti-125P5C8 antibody compositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopeschemotherapeutic agents, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to maytansinoids, ytrium,bismuth ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidiumbromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin,Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin Achain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin,enomycin, curicin, crotin, calicheamicin, sapaonaria officinalisinhibitor, and glucocorticoid and other chemotherapeutic agents, as wellas radioisotopes such as At²¹¹, I^(131, I) ¹²⁵, Y⁹⁰, Re¹⁸⁶,Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³² and radioactive isotopes of Lu. Antibodies may also beconjugated to an anti-cancer pro-drug activating enzyme capable ofconverting the pro-drug to its active form.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37 degrees C. and temperatures for washingin 0.1×SSC/0.1% SDS are above 55 degrees C.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides that correspondor are complementary to genes other than the 125P5C8 gene or that encodepolypeptides other than 125P5C8 gene product or fragments thereof. Askilled artisan can readily employ nucleic acid isolation procedures toobtain an isolated 125P5C8 polynucleotide.

As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the 125P5C8protein from cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated 125P5C8 protein. Alternatively, anisolated protein can be prepared by chemical means.

The term “mammal” as used herein refers to any organism classified as amammal, including mice, rats, rabbits, dogs, cats, cows, horses andhumans. In one embodiment of the invention, the mammal is a mouse. Inanother embodiment of the invention, the mammal is a human.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers that have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage TxNxM+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is a preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation. Approximately half of theseandrogen-refractory patients die within 6 months after developing thatstatus. The most common site for prostate cancer metastasis is bone.Prostate cancer bone metastases are often osteoblastic rather thanosteolytic (i.e., resulting in net bone formation). Bone metastases arefound most frequently in the spine, followed by the femur, pelvis, ribcage, skull and humerus. Other common sites for metastasis include lymphnodes, lung, liver and brain. Metastatic prostate cancer is typicallydiagnosed by open or laparoscopic pelvic lymphadenectomy, whole bodyradionuclide scans, skeletal radiography, and/or bone lesion biopsy.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the antibodies comprising the population are identical except forpossible naturally occurring mutations that are present in minoramounts.

As used herein “motif” as in biological motif of an 125P5C8-relatedprotein, refers to any pattern of amino acids forming part of theprimary sequence of a protein, that is associated with a particularfunction (e.g. protein-protein interaction, protein-DNA interaction,etc) or modification (e.g. that is phosphorylated, glycosylated oramidated), or localization (e.g. secretory sequence, nuclearlocalization sequence, etc.) or a sequence that is correlated with beingimmunogenic, either humorally or cellularly. A motif can be eithercontiguous or capable of being aligned to certain positions that aregenerally correlated with a certain function or property.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA and/or RNA. In the art, this term if often used interchangeably with“oligonucleotide”. A polynucleotide can comprise a nucleotide sequencedisclosed herein wherein thymidine (T) (as shown for example in SEQ IDNO: 1) can also be uracil (U); this definition pertains to thedifferences between the chemical structures of DNA and RNA, inparticular the observation that one of the four major bases in RNA isuracil (U) instead of thymidine (T).

As used herein, the term “polypeptide” means a polymer of at least about4, 5, 6, 7, or 8 amino acids. Throughout the specification, standardthree letter or single letter designations for amino acids are used. Inthe art, this term is often used interchangeably with “peptide” or“protein”.

As used herein, a “recombinant” DNA or RNA molecule is a DNA or RNAmolecule that has been subjected to molecular manipulation in vitro.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 125P5C8 protein shown in FIG. 2). An analogis an example of a variant protein.

As used herein, the 125P5C8-related gene and 125P5C8-related proteinincludes the 125P5C8 genes and proteins specifically described herein,as well as structurally and/or functionally similar variants or analogof the foregoing. 125P5C8 peptide analogs generally share at least about50%, 60%, 70%, 80%, 90% or more amino acid homology (using BLASTcriteria). 125P5C8 nucleotide analogs preferably share 50%, 60%, 70%,80%, 90% or more nucleic acid homology (using BLAST criteria). In someembodiments, however, lower homology is preferred so as to selectpreferred residues in view of species-specific codon preferences and/oroptimal peptide epitopes tailored to a particular target population, asis appreciated by those skilled in the art.

The 125P5C8-related proteins of the invention include those specificallyidentified herein, as well as allelic variants, conservativesubstitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 125P5C8 proteins orfragments thereof, as well as fusion proteins of a 125P5C8 protein and aheterologous polypeptide are also included. Such 125P5C8 proteins arecollectively referred to as the 125P5C8-related proteins, the proteinsof the invention, or 125P5C8. As used herein, the term “125P5C8-relatedprotein” refers to a polypeptide fragment or an 125P5C8 protein sequenceof 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45,50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 amino acids.

II.) Properties of 125P5C8.

As disclosed herein, 125P5C8 exhibits specific properties that areanalogous to those found in a family of molecules whose polynucleotides,polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells(HTL) and anti-polypeptide antibodies are used in well known diagnosticassays that examine conditions associated with dysregulated cell growthsuch as cancer, in particular prostate cancer (see, e.g., both itshighly specific pattern of tissue expression as well as itsoverexpression in prostate cancers as described for example in Example4). The best-known member of this class is PSA, the archetypal markerthat has been used by medical practitioners for years to identify andmonitor the presence of prostate cancer (see, e.g., Merrill et al., J.Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. Aug;162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19):1635-1640(1999)). A variety of other diagnostic markers are also used inthis context including p53 and K-ras (see, e.g., Tulchinsky et al., IntJ Mol Med Jul. 4, 1999 (1):99-102 and Minimoto et al., Cancer DetectPrev 2000;24(1):1-12). Therefore, this disclosure of the 125P5C8polynucleotides and polypeptides (as well as the 125P5C8 polynucleotideprobes and anti-125P5C8 antibodies used to identify the presence ofthese molecules) and their properties allows skilled artisans to utilizethese molecules in methods that are analogous to those used, forexample, in a variety of diagnostic assays directed to examiningconditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 125P5C8polynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays whichemploy, e.g., PSA polynucleotides, polypeptides, reactive T cells andantibodies. For example, just as PSA polynucleotides are used as probes(for example in Northern analysis, see, e.g., Sharief et al., Biochem.Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCRanalysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000))to observe the presence and/or the level of PSA mRNAs in methods ofmonitoring PSA overexpression or the metastasis of prostate cancers, the125P5C8 polynucleotides described herein can be utilized in the same wayto detect 125P5C8 overexpression or the metastasis of prostate and othercancers expressing this gene. Alternatively, just as PSA polypeptidesare used to generate antibodies specific for PSA which can then be usedto observe the presence and/or the level of PSA proteins in methods tomonitor PSA protein overexpression (see, e.g., Stephan et al., Urology55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g.,Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 125P5C8polypeptides described herein can be utilized to generate antibodies foruse in detecting 125P5C8 overexpression or the metastasis of prostatecells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 125P5C8polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 125P5C8-expressing cells (lymph node) is found tocontain 125P5C8-expressing cells such as the 125P5C8 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 125P5C8 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 125P5C8 or express 125P5C8 at adifferent level are found to express 125P5C8 or have an increasedexpression of 125P5C8 (see, e.g., the 125P5C8 expression in kidney, lungand colon cancer cells and in patient samples etc. shown in FIGS. 5-9).In such assays, artisans may further wish to generate supplementaryevidence of metastasis by testing the biological sample for the presenceof a second tissue restricted marker (in addition to 125P5C8) such asPSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,125P5C8 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in Example 4, where a 125P5C8 polynucleotidefragment is used as a probe to show the expression of 125P5C8 RNAs incancer cells. In addition, variant polynucleotide sequences aretypically used as primers and probes for the corresponding mRNAs in PCRand Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther.Nov.-Dec. 11, 1996 (6):407-13 and Current Protocols In MolecularBiology, Volume 2, Unit 2, Frederick M. Ausubul et al. eds., 1995)).Polynucleotide fragments and variants are useful in this context wherethey are capable of binding to a target polynucleotide sequence (e.g.the 125P5C8 polynucleotide shown in SEQ ID NO: 1) under conditions ofhigh stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 125P5C8 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubulet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 125P5C8biological motifs discussed herein or available in the art. Polypeptidefragments, variants or analogs are typically useful in this context aslong as they comprise an epitope capable of generating an antibody or Tcell specific for a target polypeptide sequence (e.g. the 125P5C8polypeptide shown in SEQ ID NO: 2).

As shown herein, the 125P5C8 polynucleotides and polypeptides (as wellas the 125P5C8 polynucleotide probes and anti-125P5C8 antibodies or Tcells used to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers of the prostate.Diagnostic assays that measure the presence of 125P5C8 gene products, inorder to evaluate the presence or onset of a disease condition describedherein, such as prostate cancer, are used to identify patients forpreventive measures or further monitoring, as has been done sosuccessfully with PSA. Moreover, these materials satisfy a need in theart for molecules having similar or complementary characteristics to PSAin situations where, for example, a definite diagnosis of metastasis ofprostatic origin cannot be made on the basis of a test for PSA alone(see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)),and consequently, materials such as 125P5C8 polynucleotides andpolypeptides (as well as the 125P5C8 polynucleotide probes andanti-125P5C8 antibodies used to identify the presence of thesemolecules) must be employed to confirm metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 125P5C8polynucleotides disclosed herein have a number of other specificutilities such as their use in the identification of oncogeneticassociated chromosomal abnormalities in the chromosomal region to whichthe 125P5C8 gene maps (see Example 3 below). Moreover, in addition totheir use in diagnostic assays, the 125P5C8-related proteins andpolynucleotides disclosed herein have other utilities such as their usein the forensic analysis of tissues of unknown origin (see, e.g.,Takahama K Forensic Sci Int Jun. 28, 1996; 80(1-2): 63-9).

Additionally, 125P5C8-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 125P5C8. For example, the amino acid or nucleicacid sequence of FIG. 2, or fragments thereof, can be used to generatean immune response to the 125P5C8 antigen. Antibodies or other moleculesthat react with 125P5C8 can be used to modulate the function of thismolecule, and thereby provide a therapeutic benefit.

III.) 125P5C8 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of an 125P5C8 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding an 125P5C8-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to an 125P5C8 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toan 125P5C8 gene, mRNA, or to an 125P5C8 encoding polynucleotide(collectively, “125P5C8 polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 125P5C8 polynucleotide include: a 125P5C8polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 125P5C8 as shown in FIG. 2, wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. Further 125P5C8nucleotides comprise, where T can be U:

(a) a polynucleotide having the sequence as shown in FIG. 2 (SEQ ID NO:1), from nucleotide residue number 1 through nucleotide residue number2103; or,

(b) a polynucleotide having the sequence as shown in FIG. 2 (SEQ ID NO:1), from nucleotide residue number 1 through nucleotide residue number2100; or,

(c) a polynucleotide having the sequence as shown in FIG. 2 (SEQ ID NO:1), from nucleotide residue number 1 through nucleotide residue number2097; or

(d) a polynucleotide of at least 10 bases of FIG. 2 (SEQ ID NO: 1) thatcomprises the base at position 339;

(e) a polynucleotide of at least 10 bases of FIG. 2 (SEQ ID NO: 1) thatcomprises the base at position 1119;

(f) a polynucleotide of at least 10 bases of FIG. 2 (SEQ ID NO: 1) thatcomprises the base at position 2065;

(g) a polynucleotide that selectively hybridizes under stringentconditions to a polynucleotide of (a)-(f).

As used herein, a range is understood to specifically disclose all wholeunit positions thereof. Moreover, a peptide that is encoded by any ofthe foregoing is also within the scope of the invention. An alternativeembodiment comprises a polynucleotide of the invention with a provisothat the nucleic acid does not include one or more of the specifiedpositions or ranges.

Also within the scope of the invention is a nucleotide, as well as anypeptide encoded thereby, that starts at any of the following positionsand ends at a higher position or range: 1, 1-338, 339, 340-1118, 1119,1120-2064, 2065, 2066-2097, 2100, and 2103; wherein a range as used inthis section is understood to specifically disclose all whole unitpositions thereof.

Another embodiment of the invention comprises a polynucleotide thatencodes a 125P5C8-related protein whose sequence is encoded by the cDNAcontained in the plasmid deposited with American Type Culture Collection(ATCC) as Accession No. [***]. Another embodiment comprises apolynucleotide that hybridizes under stringent hybridization conditions,to the human 125P5C8 cDNA shown in FIG. 2 or to a polynucleotidefragment thereof.

Typical embodiments of the invention disclosed herein include 125P5C8polynucleotides that encode specific portions of the 125P5C8 mRNAsequence (and those which are complementary to such sequences) such asthose that encode the protein and fragments thereof, for example of 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 125P5C8protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 125P5C8 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 20 to about amino acid30 of the 125P5C8 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 30 to about amino acid 40 of the 125P5C8protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 40 to about amino acid 50 of the 125P5C8 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 50 to about amino acid60 of the 125P5C8 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 60 to about amino acid 70 of the 125P5C8protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 70 to about amino acid 80 of the 125P5C8 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 80 to about amino acid90 of the 125P5C8 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 90 to about amino acid 100 of the 125P5C8protein shown in FIG. 2 or FIG. 3, in increments of about 10 aminoacids, ending at the carboxyl terminal amino acid set forth in FIG. 2 orFIG. 3. Accordingly polynucleotides encoding portions of the amino acidsequence (of about 10 amino acids), of amino acids 100 through thecarboxyl terminal amino acid of the 125P5C8 protein are embodiments ofthe invention. Wherein it is understood that each particular amino acidposition discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of the 125P5C8 proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the 125P5C8 protein shown in FIG.2 can be generated by a variety of techniques well known in the art.These polynucleotide fragments can include any portion of the 125P5C8sequence as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed hereininclude 125P5C8 polynucleotide fragments encoding one or more of thebiological motifs contained within the 125P5C8 protein sequence,including one or more of the motif-bearing subsequences of the 125P5C8protein set forth in Tables V-XIX. In another embodiment, typicalpolynucleotide fragments of the invention encode one or more of theregions of 125P5C8 that exhibit homology to a known molecule. In anotherembodiment of the invention, typical polynucleotide fragments can encodeone or more of the 125P5C8 N-glycosylation sites, cAMP andcGMP-dependent protein kinase phosphorylation sites, casein kinase IIphosphorylation sites or N-myristoylation site and amidation sites.

III.A.) Uses of 125P5C8 Polynucleotides III.A.1.) Monitoring of GeneticAbnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 125P5C8 gene maps to the chromosomallocation set forth in Example 3). For example, because the 125P5C8 genemaps to this chromosome, polynucleotides that encode different regionsof the 125P5C8 protein are used to characterize cytogeneticabnormalities of this chromosomal locale, such as abnormalities that areidentified as being associated with various cancers. In certain genes, avariety of chromosomal abnormalities including rearrangements have beenidentified as frequent cytogenetic abnormalities in a number ofdifferent cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4):81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) andFinger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotidesencoding specific regions of the 125P5C8 protein provide new tools thatcan be used to delineate, with greater precision than previouslypossible, cytogenetic abnormalities in the chromosomal region thatencodes 125P5C8 that may contribute to the malignant phenotype. In thiscontext, these polynucleotides satisfy a need in the art for expandingthe sensitivity of chromosomal screening in order to identify moresubtle and less common chromosomal abnormalities (see e.g. Evans et al.,Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 125P5C8 was shown to be highly expressed in prostate andother cancers, 125P5C8 polynucleotides are used in methods assessing thestatus of 125P5C8 gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the 125P5C8protein are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 125P5C8 gene, suchas such regions containing one or more motifs. Exemplary assays includeboth RT-PCR assays as well as single-strand conformation polymorphism(SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8):369-378 (1999), both of which utilize polynucleotides encoding specificregions of a protein to examine these regions within the protein.

III.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 125P5C8. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 125P5C8 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,125P5C8. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 125P5C8 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990); andIyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional125P5C8 antisense oligonucleotides of the present invention includemorpholino antisense oligonucleotides known in the art (see, e.g.,Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6:169-175).

The 125P5C8 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3”codons of the125P5C8 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 125P5C8 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 125P5C8 antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 125P5C8 mRNA. Optionally, 125P5C8antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 125P5C8. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 125P5C8 expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

III.A.3.) Primers and Primer Pairs

Further specific embodiments of this nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 125P5C8 polynucleotide in a sample and as ameans for detecting a cell expressing a 125P5C8 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 125P5C8 cDNA sequences shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 125P5C8 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 125P5C8 mRNA.

The 125P5C8 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 125P5C8 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 125P5C8 polypeptides; as tools formodulating or inhibiting the expression of the 125P5C8 gene(s) and/ortranslation of the 125P5C8 transcript(s); and as therapeutic agents.

III.A.4.) Isolation of 125P5C8-Encoding Nucleic Acid Molecules

The 125P5C8 cDNA sequences described herein enable the isolation ofother polynucleotides encoding 125P5C8 gene product(s), as well as theisolation of polynucleotides encoding 125P5C8 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms ofthe 125P5C8 gene product as well as polynucleotides that encode analogsof 125P5C8-related proteins. Various molecular cloning methods that canbe employed to isolate full length cDNAs encoding an 125P5C8 gene arewell known (see, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 125P5C8gene cDNAs can be identified by probing with a labeled 125P5C8 cDNA or afragment thereof. For example, in one embodiment, the 125P5C8 cDNA (FIG.2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a 125P5C8gene. The 125P5C8 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 125P5C8 DNAprobes or primers.

III.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containingan 125P5C8 polynucleotide, fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook et al, 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 125P5C8 polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of 125P5C8or a fragment, analog or homolog thereof can be used to generate 125P5C8proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of125P5C8 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRatkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 125P5C8 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 125P5C8 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 125P5C8 and 125P5C8 mutations or analogs.

Recombinant human 125P5C8 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 125P5C8-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 125P5C8 or fragment,analog or homolog thereof, the 125P5C8 or related protein is expressedin the 293T cells, and the recombinant 125P5C8 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-125P5C8 antibodies). In another embodiment, a 125P5C8 codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish 125P5C8 expressing cell lines. Various otherexpression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to the125P5C8 coding sequence can be used for the generation of a secretedform of recombinant 125P5C8 protein.

As discussed herein, redundancy in the genetic code permits variation in125P5C8 gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET such as:http://www.dna.affrc.go.jp/˜nakamura/codon.html.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

IV.) 125P5C8-Related Proteins

Another aspect of the present invention provides 125P5C8-relatedproteins. Specific embodiments of 125P5C8 proteins comprise apolypeptide having all or part of the amino acid sequence of human125P5C8 as shown in FIG. 2. Alternatively, embodiments of 125P5C8proteins comprise variant, homolog or analog polypeptides that havealterations in the amino acid sequence of 125P5C8 shown in FIG. 2.

In general, naturally occurring allelic variants of human 125P5C8 sharea high degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of the 125P5C8 protein containconservative amino acid substitutions within the 125P5C8 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 125P5C8. One class of 125P5C8allelic variants are proteins that share a high degree of homology withat least a small region of a particular 125P5C8 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15conservative substitutions. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g. Table III herein; pages 13-15 “Biochemistry”2^(nd) ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS1992 Vol 89 10915-10919; Lei et al., J Biol Chem May 19, 1995;270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 125P5C8 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.125P5C8 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 125P5C8 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 125P5C8 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 125P5C8 protein having the amino acid sequence of SEQID NO: 2. As used in this sentence, “cross reactive” means that anantibody or T cell that specifically binds to an 125P5C8 variant alsospecifically binds to the 125P5C8 protein having the amino acid sequenceof SEQ ID NO: 2. A polypeptide ceases to be a variant of the proteinshown in SEQ ID NO: 2 when it no longer contains any epitope capable ofbeing recognized by an antibody or T cell that specifically binds to the125P5C8 protein. Those skilled in the art understand that antibodiesthat recognize proteins bind to epitopes of varying size, and a groupingof the order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al.,Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985)135(4):2598-608.

Another class of 125P5C8-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with the amino acid sequence of SEQ ID NO:2 or a fragment thereof. Another specific class of 125P5C8 proteinvariants or analogs comprise one or more of the 125P5C8 biologicalmotifs described herein or presently known in the art. Thus, encompassedby the present invention are analogs of 125P5C8 fragments (nucleic oramino acid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the 532 amino acid sequence of the125P5C8 protein shown in FIG. 2. For example, representative embodimentsof the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more contiguous amino acids of the 125P5C8protein shown in FIG. 2 (SEQ ID NO: 2).

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of the 125P5C8 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of the 125P5C8protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of the 125P5C8 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 toabout amino acid 40 of the 125P5C8 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofthe 125P5C8 protein shown in FIG. 2 or FIG. 3, polypeptides consistingof about amino acid 50 to about amino acid 60 of the 125P5C8 proteinshown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid60 to about amino acid 70 of the 125P5C8 protein shown in FIG. 2 or FIG.3, polypeptides consisting of about amino acid 70 to about amino acid 80of the 125P5C8 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 80 to about amino acid 90 of the 125P5C8protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 90 to about amino acid 100 of the 125P5C8 protein shown inFIG. 2 or FIG. 3, etc. throughout the entirety of the 125P5C8 amino acidsequence. Moreover, polypeptides consisting of about amino acid 1 (or 20or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.)of the 125P5C8 protein shown in FIG. 2 are embodiments of the invention.It is to be appreciated that the starting and stopping positions in thisparagraph refer to the specified position as well as that position plusor minus 5 residues.

125P5C8-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a 125P5C8-related protein. In one embodiment,nucleic acid molecules provide a means to generate defined fragments ofthe 125P5C8 protein (or variants, homologs or analogs thereof).

IV.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 125P5C8 polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within the 125P5C8polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs areknown in the art, and a protein can be evaluated for the presence ofsuch motifs by a number of publicly available sites (see, e.g.:http://pfam.wustl.edu/;http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.htmlhttp://psort.ims.u-tokyo.ac.jp/; http://www.cbs.dtu.dk/;http//www.ebi.ac.uk/interpro/scan.html;http://www.expasy.ch/tools/scnpsit1.html; Epimatrix™ and Epimer™, BrownUniversity,http://www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; andBIMAS, http://bimas.dcrt.nih.gov/.). Motif bearing subsequences of the125P5C8 protein are set forth and identified in Table XIX.

Table XX sets forth several frequently occurring motifs based on pfamsearches (http://pfam.wustl.edu/). The columns of Table XX list (1)motif name abbreviation, (2) percent identity found amongst thedifferent member of the motif family, (3) motif name or description and(4) most common function; location information is included if the motifis relevant for location.

Polypeptides comprising one or more of the 125P5C8 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 125P5C8 motifsdiscussed above are associated with growth dysregulation and because125P5C8 is overexpressed in certain cancers (See, e.g., Table I). Caseinkinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C,for example, are enzymes known to be associated with the development ofthe malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995);hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterzielet al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2):305-309 (1998)). Moreover, both glycosylation and myristoylation areprotein modifications also associated with cancer and cancer progression(see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999);Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation isanother protein modification also associated with cancer and cancerprogression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr.(13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables V-XVIII. CTL epitopescan be determined using specific algorithms to identify peptides withinan 125P5C8 protein that are capable of optimally binding to specifiedHLA alleles (e.g., Table IV (A) and Table IV (B); Epimatrix™ andEpimer™, Brown University,http://www.brow.edu/Research/TB-HIV_Lab/epimatirx/epimatrix.html; andBIMAS, http://bimas.dcrt.nih.gov/. Moreover, processes for identifyingpeptides that have sufficient binding affinity for HLA molecules andwhich are correlated with being immunogenic epitopes, are well known inthe art, and are carried out without undue experimentation. In addition,processes for identifying peptides that are immunogenic epitopes, arewell known in the art, and are carried out without undue experimentationeither in vitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI motifs or Table IV (A) and the HTL motif of Table IV (B)). The epitopeis analoged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 9733602 to Chesnut et al.; Sette,Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondoet al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol.1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt etal., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7(1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J.Immunol. 1991 147(8):2663-2669; Alexander et al., Immunity 1994 1(9):751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the inventions include polypeptides comprisingcombinations of the different motifs set forth in Table XIX, and/or, oneor more of the predicted CTL epitopes of Table V through Table XVIII,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typicallythe number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

125P5C8-related proteins are embodied in many forms, preferably inisolated form. A purified 125P5C8 protein molecule will be substantiallyfree of other proteins or molecules that impair the binding of 125P5C8to antibody, T cell or other ligand. The nature and degree of isolationand purification will depend on the intended use. Embodiments of a125P5C8-related proteins include purified 125P5C8-related proteins andfunctional, soluble 125P5C8-related proteins. In one embodiment, afunctional, soluble 125P5C8 protein or fragment thereof retains theability to be bound by antibody, T cell or other ligand.

The invention also provides 125P5C8 proteins comprising biologicallyactive fragments of the 125P5C8 amino acid sequence shown in FIG. 2.Such proteins exhibit properties of the 125P5C8 protein, such as theability to elicit the generation of antibodies that specifically bind anepitope associated with the 125P5C8 protein; to be bound by suchantibodies; to elicit the activation of HTL or CTL; and/or, to berecognized by HTL or CTL.

125P5C8-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or on the basis of immunogenicity. Fragmentsthat contain such structures are particularly useful in generatingsubunit-specific anti-125P5C8 antibodies, or T cells or in identifyingcellular factors that bind to 125P5C8.

CTL epitopes can be determined using specific algorithms to identifypeptides within an 125P5C8 protein that are capable of optimally bindingto specified HLA alleles (e.g., Table IV (A) and Table IV (B);Epimatrix™ and Epimer™, Brown University(http://www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); andBIMAS, http:/bimas.dcrt.nih.gov/). Illustrating this, peptide epitopesfrom 125P5C8 that are presented in the context of human MHC class Imolecules HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (TablesV-XVIII). Specifically, the complete amino acid sequence of the 125P5C8protein was entered into the HLA Peptide Motif Search algorithm found inthe Bioinformatics and Molecular Analysis Section (BIMAS) web sitelisted above. The HLA peptide motif search algorithm was developed byDr. Ken Parker based on binding of specific peptide sequences in thegroove of HLA Class I molecules and specifically HLA-A2 (see, e.g., Falket al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3(1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J.Immunol. 152:163-75 (1994)). This algorithm allows location and rankingof 8-mer, 9-mer, and 10-mer peptides from a complete protein sequencefor predicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7(1992)). Selected results of 125P5C8 predicted binding peptides areshown in Tables V-XVIII herein. In Tables V-XVIII, the top 50 rankingcandidates, 9-mers and 10-mers, for each family member are shown alongwith their location, the amino acid sequence of each specific peptide,and an estimated binding score. The binding score corresponds to theestimated half-time of dissociation of complexes containing the peptideat 37° C. at pH 6.5. Peptides with the highest binding score arepredicted to be the most tightly bound to HLA Class I on the cellsurface for the greatest period of time and thus represent the bestimmunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or class Imotifs available in the art or which become part of the art such as setforth in Table IV (A) and Table IV (B) are to be “applied” to the125P5C8 protein. As used in this context “applied” means that the125P5C8 protein is evaluated, e.g., visually or by computer-basedpatterns finding methods, as appreciated by those of skill in therelevant art. Every subsequence of the 125P5C8 of 8, 9, 10, or 11 aminoacid residues that bears an HLA Class I motif, or a subsequence of 9 ormore amino acid residues that bear an HLA Class II motif are within thescope of the invention.

IV.B.) Expression of 125P5C8-Related Proteins

In an embodiment described in the examples that follow, 125P5C8 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 125P5C8 with a C-terminal 6XHis and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 125P5C8 protein intransfected cells. The secreted HIS-tagged 125P5C8 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

IV.C.) Modifications of 125P5C8-Related Proteins

Modifications of 125P5C8-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a 125P5C8polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthe 125P5C8. Another type of covalent modification of the 125P5C8polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.Another type of covalent modification of 125P5C8 comprises linking the125P5C8 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 125P5C8-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 125P5C8 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments ofthe 125P5C8 sequence (amino or nucleic acid) such that a molecule iscreated that is not, through its length, directly homologous to theamino or nucleic acid sequences respectively of FIG. 2. Such a chimericmolecule can comprise multiples of the same subsequence of 125P5C8. Achimeric molecule can comprise a fusion of a 125P5C8-related proteinwith a polyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind, with cytokines or with growthfactors. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the 125P5C8. In an alternative embodiment, thechimeric molecule can comprise a fusion of a 125P5C8-related proteinwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a 125P5C8polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of anIgGI molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

IV.D.) Uses of 125P5C8-Related Proteins

The proteins of the invention have a number of different specific uses.As 125P5C8 is highly expressed in prostate and other cancers,125P5C8-related proteins are used in methods that assess the status of125P5C8 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of the 125P5C8 protein are used to assess the presenceof perturbations (such as deletions, insertions, point mutations etc.)in those regions (such as regions containing one or more motifs).Exemplary assays utilize antibodies or T cells targeting 125P5C8-relatedproteins comprising the amino acid residues of one or more of thebiological motifs contained within the 125P5C8 polypeptide sequence inorder to evaluate the characteristics of this region in normal versuscancerous tissues or to elicit an immune response to the epitope.Alternatively, 125P5C8-related proteins that contain the amino acidresidues of one or more of the biological motifs in the 125P5C8 proteinare used to screen for factors that interact with that region of125P5C8.

125P5C8 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of an125P5C8 protein), for identifying agents or cellular factors that bindto 125P5C8 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 125P5C8 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to an 125P5C8 gene product.Antibodies raised against an 125P5C8 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 125P5C8protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 125P5C8-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

Various immunological assays useful for the detection of 125P5C8proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 125P5C8-expressingcells (e.g., in radioscintigraphic imaging methods). 125P5C8 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

V.) 125P5C8 Antibodies

Another aspect of the invention provides antibodies that bind to125P5C8-related proteins. Preferred antibodies specifically bind to a125P5C8-related protein and do not bind (or bind weakly) to peptides orproteins that are not 125P5C8-related proteins. For example, antibodiesbind 125P5C8 can bind 125P5C8-related proteins such as the homologs oranalogs thereof.

125P5C8 antibodies of the invention are particularly useful in prostatecancer diagnostic and prognostic assays, and imaging methodologies.Similarly, such antibodies are useful in the treatment, diagnosis,and/or prognosis of other cancers, to the extent 125P5C8 is alsoexpressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of125P5C8 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 125P5C8 and mutant 125P5C8-relatedproteins. Such assays can comprise one or more 125P5C8 antibodiescapable of recognizing and binding a 125P5C8-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 125P5C8 are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 125P5C8 antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 125P5C8 expressingcancers such as prostate cancer.

125P5C8 antibodies are also used in methods for purifying a125P5C8-related protein and for isolating 125P5C8 homologues and relatedmolecules. For example, a method of purifying a 125P5C8-related proteincomprises incubating an 125P5C8 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing a125P5C8-related protein under conditions that permit the 125P5C8antibody to bind to the 125P5C8-related protein; washing the solidmatrix to eliminate impurities; and eluting the 125P5C8-related proteinfrom the coupled antibody. Other uses of the 125P5C8 antibodies of theinvention include generating anti-idiotypic antibodies that mimic the125P5C8 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 125P5C8-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 125P5C8 canalso be used, such as a 125P5C8 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogento generate appropriate antibodies. In another embodiment, a125P5C8-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 125P5C8-related protein or 125P5C8 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of 125P5C8 as shown in FIG. 2 or FIG. 3 can beanalyzed to select specific regions of the 125P5C8 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of the 125P5C8 amino acid sequence are used to identifyhydrophilic regions in the 125P5C8 structure. Regions of the 125P5C8protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Thus, each region identifiedby any of these programs or methods is within the scope of the presentinvention. Methods for the generation of 125P5C8 antibodies are furtherillustrated by way of the examples provided herein. Methods forpreparing a protein or polypeptide for use as an immunogen are wellknown in the art. Also well known in the art are methods for preparingimmunogenic conjugates of a protein with a carrier, such as BSA, KLH orother carrier protein. In some circumstances, direct conjugation using,for example, carbodiimide reagents are used; in other instances linkingreagents such as those supplied by Pierce Chemical Co., Rockford, Ill.,are effective. Administration of a 125P5C8 immunogen is often conductedby injection over a suitable time period and with use of a suitableadjuvant, as is understood in the art. During the immunization schedule,titers of antibodies can be taken to determine adequacy of antibodyformation.

125P5C8 monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 125P5C8-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof the 125P5C8 protein can also be produced in the context of chimericor complementarity determining region (CDR) grafted antibodies ofmultiple species origin. Humanized or human 125P5C8 antibodies can alsobe produced, and are preferred for use in therapeutic contexts. Methodsfor humanizing murine and other non-human antibodies, by substitutingone or more of the non-human antibody CDRs for corresponding humanantibody sequences, are well known (see for example, Jones et al., 1986,Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327;Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter etal., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J.Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). Fully human 125P5C8 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully human125P5C8 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO98/24893, Kucherlapati and Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S.Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598issued 5 Sep. 2000). This method avoids the in vitro manipulationrequired with phage display technology and efficiently produces highaffinity authentic human antibodies.

Reactivity of 125P5C8 antibodies with an 125P5C8-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,125P5C8-related proteins, 125P5C8-expressing cells or extracts thereof.A 125P5C8 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more125P5C8 epitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

VI.) 125P5C8 Transgenic Animals

Nucleic acids that encode a 125P5C8-related protein can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. In accordance with established techniques, cDNAencoding 125P5C8 can be used to clone genomic DNA that encodes 125P5C8.The cloned genomic sequences can then be used to generate transgenicanimals containing cells that express DNA that encode 125P5C8. Methodsfor generating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat.No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would betargeted for 125P5C8 transgene incorporation with tissue-specificenhancers.

Transgenic animals that include a copy of a transgene encoding 125P5C8can be used to examine the effect of increased expression of DNA thatencodes 125P5C8. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this aspect ofthe invention, an animal is treated with a reagent and a reducedincidence of a pathological condition, compared to untreated animalsthat bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 125P5C8 can be used to constructa 125P5C8 “knock out” animal that has a defective or altered geneencoding 125P5C8 as a result of homologous recombination between theendogenous gene encoding 125P5C8 and altered genomic DNA encoding125P5C8 introduced into an embryonic cell of the animal. For example,cDNA that encodes 125P5C8 can be used to clone genomic DNA encoding125P5C8 in accordance with established techniques. A portion of thegenomic DNA encoding 125P5C8 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of the 125P5C8 polypeptide.

VII.) Methods for the Detection of 125P5C8

Another aspect of the present invention relates to methods for detecting125P5C8 polynucleotides and 125P5C8-related proteins, as well as methodsfor identifying a cell that expresses 125P5C8. The expression profile of125P5C8 makes it a diagnostic marker for metastasized disease.Accordingly, the status of 125P5C8 gene products provides informationuseful for predicting a variety of factors including susceptibility toadvanced stage disease, rate of progression, and/or tumoraggressiveness. As discussed in detail herein, the status of 125P5C8gene products in patient samples can be analyzed by a variety protocolsthat are well known in the art including immunohistochemical analysis,the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of125P5C8 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 125P5C8 polynucleotides include, for example, a 125P5C8gene or fragment thereof, 125P5C8 mRNA, alternative splice variant125P5C8 mRNAs, and recombinant DNA or RNA molecules that contain a125P5C8 polynucleotide. A number of methods for amplifying and/ordetecting the presence of 125P5C8 polynucleotides are well known in theart and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting an 125P5C8 mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing an 125P5C8 polynucleotides as sense and antisense primers toamplify 125P5C8 cDNAs therein; and detecting the presence of theamplified 125P5C8 cDNA. Optionally, the sequence of the amplified125P5C8 cDNA can be determined.

In another embodiment, a method of detecting a 125P5C8 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 125P5C8 polynucleotides assense and antisense primers; and detecting the presence of the amplified125P5C8 gene. Any number of appropriate sense and antisense probecombinations can be designed from the nucleotide sequence provided forthe 125P5C8 (FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of an125P5C8 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 125P5C8-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 125P5C8-related proteinin a biological sample comprises first contacting the sample with a125P5C8 antibody, a 125P5C8-reactive fragment thereof, or a recombinantprotein containing an antigen binding region of a 125P5C8 antibody; andthen detecting the binding of 125P5C8-related protein in the sample.

Methods for identifying a cell that expresses 125P5C8 are also withinthe scope of the invention. In one embodiment, an assay for identifyinga cell that expresses a 125P5C8 gene comprises detecting the presence of125P5C8 mRNA in the cell. Methods for the detection of particular mRNAsin cells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled 125P5C8 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for 125P5C8, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Alternatively, an assay for identifying a cell that expressesa 125P5C8 gene comprises detecting the presence of 125P5C8-relatedprotein in the cell or secreted by the cell. Various methods for thedetection of proteins are well known in the art and are employed for thedetection of 125P5C8-related proteins and cells that express125P5C8-related proteins.

125P5C8 expression analysis is also useful as a tool for identifying andevaluating agents that modulate 125P5C8 gene expression. For example,125P5C8 expression is significantly upregulated in prostate cancer, andis expressed in cancers of the tissues listed in Table I. Identificationof a molecule or biological agent that inhibits 125P5C8 expression orover-expression in cancer cells is of therapeutic value. For example,such an agent can be identified by using a screen that quantifies125P5C8 expression by RT-PCR, nucleic acid hybridization or antibodybinding.

VIII.) Methods for Monitoring the Status of 125P5C8-Related Genes andTheir Products

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant125P5C8 expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 125P5C8 in abiological sample of interest can be compared, for example, to thestatus of 125P5C8 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not effectedby a pathology). An alteration in the status of 125P5C8 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not effected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol.Dec. 9, 1996; 376(2):306-14 and U.S. Pat. No. 5,837,501) to compare125P5C8 status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 125P5C8 expressing cells) as well as the, level, andbiological activity of expressed gene products (such as 125P5C8 mRNApolynucleotides and polypeptides). Typically, an alteration in thestatus of 125P5C8 comprises a change in the location of 125P5C8 and/or125P5C8 expressing cells and/or an increase in 125P5C8 mRNA and/orprotein expression.

125P5C8 status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of the 125P5C8gene and gene products are found, for example in Ausubul et al. eds.,1995, Current Protocols In Molecular Biology, Units 2 (NorthernBlotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCRAnalysis). Thus, the status of 125P5C8 in a biological sample isevaluated by various methods utilized by skilled artisans including, butnot limited to genomic Southern analysis (to examine, for exampleperturbations in the 125P5C8 gene), Northern analysis and/or PCRanalysis of 125P5C8 mRNA (to examine, for example alterations in thepolynucleotide sequences or expression levels of 125P5C8 mRNAs), and,Western and/or immunohistochemical analysis (to examine, for examplealterations in polypeptide sequences, alterations in polypeptidelocalization within a sample, alterations in expression levels of125P5C8 proteins and/or associations of 125P5C8 proteins withpolypeptide binding partners). Detectable 125P5C8 polynucleotidesinclude, for example, a 125P5C8 gene or fragment thereof, 125P5C8 mRNA,alternative splice variants, 125P5C8 mRNAs, and recombinant DNA or RNAmolecules containing a 125P5C8 polynucleotide.

The expression profile of 125P5C8 makes it a diagnostic marker for localand/or metastasized disease, and provides information on the growth oroncogenic potential of a biological sample. In particular, the status of125P5C8 provides information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 125P5C8 status anddiagnosing cancers that express 125P5C8, such as cancers of the tissueslisted in Table I. For example, because 125P5C8 mRNA is so highlyexpressed in prostate and other cancers relative to normal prostatetissue, assays that evaluate the levels of 125P5C8 mRNA transcripts orproteins in a biological sample can be used to diagnose a diseaseassociated with 125P5C8 dysregulation, and can provide prognosticinformation useful in defining appropriate therapeutic options.

The expression status of 125P5C8 provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 125P5C8 inbiological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 125P5C8 in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of 125P5C8 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 125P5C8 expressing cells (e.g. those that express125P5C8 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth, for example, when 125P5C8-expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node), because such alterations in the status of125P5C8 in a biological sample are often associated with dysregulatedcellular growth. Specifically, one indicator of dysregulated cellulargrowth is the metastases of cancer cells from an organ of origin (suchas the prostate) to a different area of the body (such as a lymph node).In this context, evidence of dysregulated cellular growth is importantfor example because occult lymph node metastases can be detected in asubstantial proportion of patients with prostate cancer, and suchmetastases are associated with known predictors of disease progression(see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000); Su et al.,Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J UrolAugust 1995 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 125P5C8gene products by determining the status of 125P5C8 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 125P5C8gene products in a corresponding normal sample. The presence of aberrant125P5C8 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 125P5C8 mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 125P5C8 mRNA can, for example, beevaluated in tissue samples including but not limited to those listed inTable I. The presence of significant 125P5C8 expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express 125P5C8mRNA or express it at lower levels.

In a related embodiment, 125P5C8 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 125P5C8 protein expressed by cells ina test tissue sample and comparing the level so determined to the levelof 125P5C8 expressed in a corresponding normal sample. In oneembodiment, the presence of 125P5C8 protein is evaluated, for example,using immunohistochemical methods. 125P5C8 antibodies or bindingpartners capable of detecting 125P5C8 protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status 125P5C8 nucleotideand amino acid sequences in a biological sample in order to identifyperturbations in the structure of these molecules. These perturbationscan include insertions, deletions, substitutions and the like. Suchevaluations are useful because perturbations in the nucleotide and aminoacid sequences are observed in a large number of proteins associatedwith a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999,J. Cutan. Pathol. 26(8):369-378). For example, a mutation in thesequence of 125P5C8 may be indicative of the presence or promotion of atumor. Such assays therefore have diagnostic and predictive value wherea mutation in 125P5C8 indicates a potential loss of function or increasein tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 125P5C8 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. No. 5,382,510 issued 7, Sep. 1999, and U.S.Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of the 125P5C8 genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration ispresent in at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks et al, Cancer Epidemiol. Biomarkers Prev., 1998,7:531-536). In another example, expression of the LAGE-I tumor specificgene (which is not expressed in normal prostate but is expressed in25-50% of prostate cancers) is induced by deoxy-azacytidine inlymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). Avariety of assays for examining methylation status of a gene are wellknown in the art. For example, one can utilize, in Southernhybridization approaches, methylation-sensitive restriction enzymeswhich cannot cleave sequences that contain methylated CpG sites toassess the methylation status of CpG islands. In addition, MSP(methylation specific PCR) can rapidly profile the methylation status ofall the CpG sites present in a CpG island of a given gene. Thisprocedure involves initial modification of DNA by sodium bisulfite(which will convert all unmethylated cytosines to uracil) followed byamplification using primers specific for methylated versus unmethylatedDNA. Protocols involving methylation interference can also be found forexample in Current Protocols In Molecular Biology, Unit 12, Frederick M.Ausubul et al. eds., 1995.

Gene amplification is an additional method for assessing the status of125P5C8. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 125P5C8 expression. The presence of RT-PCRamplifiable 125P5C8 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting125P5C8 mRNA or 125P5C8 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 125P5C8 mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of 125P5C8 in prostate or other tissue isexamined, with the presence of 125P5C8 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 125P5C8 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 125P5C8 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 125P5C8 mRNA or 125P5C8 proteinexpressed by tumor cells, comparing the level so determined to the levelof 125P5C8 mRNA or 125P5C8 protein expressed in a corresponding normaltissue taken from the same individual or a normal tissue referencesample, wherein the degree of 125P5C8 mRNA or 125P5C8 protein expressionin the tumor sample relative to the normal sample indicates the degreeof aggressiveness. In a specific embodiment, aggressiveness of a tumoris evaluated by determining the extent to which 125P5C8 is expressed inthe tumor cells, with higher expression levels indicating moreaggressive tumors. Another embodiment is the evaluation of the integrityof 125P5C8 nucleotide and amino acid sequences in a biological sample,in order to identify perturbations in the structure of these moleculessuch as insertions, deletions, substitutions and the like. The presenceof one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 125P5C8 mRNA or125P5C8 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 125P5C8 mRNA or 125P5C8 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 125P5C8 mRNA or 125P5C8protein expression in the tumor sample over time provides information onthe progression of the cancer. In a specific embodiment, the progressionof a cancer is evaluated by determining 125P5C8 expression in the tumorcells over time, where increased expression over time indicates aprogression of the cancer. Also, one can evaluate the integrity 125P5C8nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 125P5C8 gene and125P5C8 gene products (or perturbations in 125P5C8 gene and 125P5C8 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSA, PSCA andPSM expression for prostate cancer etc.) as well as gross cytologicalobservations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al.,1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of 125P5C8 gene and 125P5C8 gene products (or perturbationsin 125P5C8 gene and 125P5C8 gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 125P5C8 gene and 125P5C8 gene products (or perturbationsin 125P5C8 gene and 125P5C8 gene products) and another factor associatedwith malignancy entails detecting the overexpression of 125P5C8 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample (or PSCA or PSM expression), and observing acoincidence of 125P5C8 mRNA or protein and PSA mRNA or proteinoverexpression (or PSCA or PSM expression). In a specific embodiment,the expression of 125P5C8 and PSA mRNA in prostate tissue is examined,where the coincidence of 125P5C8 and PSA mRNA overexpression in thesample indicates the existence of prostate cancer, prostate cancersusceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 125P5C8 mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 125P5C8 mRNAinclude in situ hybridization using labeled 125P5C8 riboprobes, Northernblot and related techniques using 125P5C8 polynucleotide probes, RT-PCRanalysis using primers specific for 125P5C8, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR is usedto detect and quantify 125P5C8 mRNA expression. Any number of primerscapable of amplifying 125P5C8 can be used for this purpose, includingbut not limited to the various primer sets specifically describedherein. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 125P5C8 protein can be used inan immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules that Interact with 125P5C8

The 125P5C8 protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with 125P5C8, as well as pathways activated by 125P5C8 viaany one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S.Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available inthe art for genome-based predictions of protein function (see, e.g.,Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 125P5C8 protein sequences. In such methods, peptidesthat bind to a molecule such as 125P5C8 are identified by screeninglibraries that encode a random or controlled collection of amino acids.Peptides encoded by the libraries are expressed as fusion proteins ofbacteriophage coat proteins, the bacteriophage particles are thenscreened against the protein of interest.

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 125P5C8 proteinsequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 125P5C8 are used to identifyprotein-protein interactions mediated by 125P5C8. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton BJ, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 125P5C8protein can be immunoprecipitated from 125P5C8-expressing cell linesusing anti-125P5C8 antibodies. Alternatively, antibodies against His-tagcan be used in a cell line engineered to express 125P5C8 (vectorsmentioned above). The immunoprecipitated complex can be examined forprotein association by procedures such as Western blotting,³⁵S-methionine labeling of proteins, protein microsequencing, silverstaining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 125P5C8 can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 125P5C8's ability to mediatephosphorylation and de-phosphorylation, interaction with DNA or RNAmolecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate ion channel, protein pump, or cell communication function of125P5C8 are identified and used to treat patients that have a cancerthat expresses the 125P5C8 antigen (see, e.g., Hille, B., Ionic Channelsof Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland, Mass.,1992). Moreover, ligands that regulate 125P5C8 function can beidentified based on their ability to bind 125P5C8 and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of125P5C8 and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying both activators and inhibitors of 125P5C8.

An embodiment of this invention comprises a method of screening for amolecule that interacts with an 125P5C8 amino acid sequence shown inFIG. 2 and FIG. 3 (SEQ ID NO: 2), comprising the steps of contacting apopulation of molecules with the 125P5C8 amino acid sequence, allowingthe population of molecules and the 125P5C8 amino acid sequence tointeract under conditions that facilitate an interaction, determiningthe presence of a molecule that interacts with the 125P5C8 amino acidsequence, and then separating molecules that do not interact with the125P5C8 amino acid sequence from molecules that do. In a specificembodiment, the method further comprises purifying a molecule thatinteracts with the 125P5C8 amino acid sequence. The identified moleculecan be used to modulate a function performed by 125P5C8. In a preferredembodiment, the 125P5C8 amino acid sequence is contacted with a libraryof peptides.

X.) Therapeutic Methods and Compositions

The identification of 125P5C8 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in prostate andother cancers, opens a number of therapeutic approaches to the treatmentof such cancers. As discussed herein, it is possible that 125P5C8functions as a transcription factor involved in activatingtumor-promoting genes or repressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of the125P5C8 protein are useful for patients suffering a cancer thatexpresses 125P5C8. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of the 125P5C8 protein with its binding partner or withothers proteins. Another class comprises a variety of methods forinhibiting the transcription of the 125P5C8 gene or translation of125P5C8 mRNA.

X.A.) 125P5C8 as a Target for Antibody-Based Therapy

125P5C8 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 125P5C8 is expressed by cancer cells of various lineages and notby corresponding normal cells, systemic administration of125P5C8-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 125P5C8 areuseful to treat 125P5C8-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

125P5C8 antibodies can be introduced into a patient such that theantibody binds to 125P5C8 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 125P5C8,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of the 125P5C8 sequence shown in FIG. 2. In addition,skilled artisans understand that it is routine to conjugate antibodiesto cytotoxic agents. When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 125P5C8), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-125P5C8 antibody) that binds to a marker (e.g. 125P5C8)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing 125P5C8, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to a125P5C8 epitope, and, exposing the cell to the antibody-agent conjugate.Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-125P5C8 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of Y⁹¹ or I¹³¹ toanti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. orBexxar™, Coulter Pharmaceuticals), while others involveco-administration of antibodies and other therapeutic agents, such asHerceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). To treatprostate cancer, for example, 125P5C8 antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation.

Although 125P5C8 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 125P5C8expression, preferably using immunohistochemical assessments of tumortissue, quantitative 125P5C8 imaging, or other techniques that reliablyindicate the presence and degree of 125P5C8 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-125P5C8 monoclonal antibodies that treat prostate and other cancersinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, anti-125P5C8monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-125P5C8 mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 125P5C8. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-125P5C8 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 125P5C8antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-125P5C8 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-125P5C8 mAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-125P5C8 mAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-125P5C8 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-125P5C8antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1 to about 10 mg/kg body weight. In general, doses in the range of10-500 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-125P5C8 mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose is administeredas a 30 minute or longer infusion, provided the initial dose was welltolerated. As appreciated by those of skill in the art, various factorscan influence the ideal dose regimen in a particular case. Such factorsinclude, for example, the binding affinity and half life of the Ab ormAbs used, the degree of 125P5C8 expression in the patient, the extentof circulating shed 125P5C8 antigen, the desired steady-state antibodyconcentration level, frequency of treatment, and the influence ofchemotherapeutic or other agents used in combination with the treatmentmethod of the invention, as well as the health status of a particularpatient.

Optionally, patients should be evaluated for the levels of 125P5C8 in agiven sample (e.g. the levels of circulating 125P5C8 antigen and/or125P5C8 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (such as serum PSA levels in prostate cancer therapy).

X.B.) Anti-Cancer Vaccines

The invention further provides cancer vaccines comprising a125P5C8-related protein or 125P5C8-related nucleic acid. In view of theexpression of 125P5C8, cancer vaccines prevent and/or treat125P5C8-expressing cancers without creating non-specific effects onnon-target tissues. The use of a tumor antigen in a vaccine thatgenerates humoral and/or cell-mediated immune responses as anti-cancertherapy is well known in the art and has been employed in prostatecancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995,Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol.159:3113-3117).

Genetic immunization methods can be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing 125P5C8. Constructs comprising DNA encoding a125P5C8-related protein/immunogen and appropriate regulatory sequencescan be injected directly into muscle or skin of an individual, such thatthe cells of the muscle or skin take-up the construct and express theencoded 125P5C8 protein/immunogen. Alternatively, a vaccine comprises a125P5C8-related protein. Expression of the 125P5C8-related proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against cells that bear 125P5C8 protein.Various prophylactic and therapeutic genetic immunization techniquesknown in the art can be used (for review, see information and referencespublished at Internet address www.genweb.com).

Such methods can be readily practiced by employing a 125P5C8-relatedprotein, or an 125P5C8-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 125P5C8 immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med Febrary 1999 31(1):66-78; Maruyama et al., CancerImmunol Immunother June 2000 49(3):123-32) Briefly, such methods ofgenerating an immune response (e.g. humoral and/or cell-mediated) in amammal, comprise the steps of: exposing the mammal's immune system to animmunoreactive epitope (e.g. an epitope present in the 125P5C8 proteinshown in SEQ ID NO: 2 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, the 125P5C8 immunogen contains a biological motif.

CTL epitopes can be determined using specific algorithms to identifypeptides within 125P5C8 protein that are capable of optimally binding tospecified HLA alleles (e.g., Table IV (A) and Table IV (B); Epimer™ andEpimatrix™, Brown University(http://www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html);and, BIMAS, (http://bimas.dcrt.nih.gov/). In a preferred embodiment, the125P5C8 immunogen contains one or more amino acid sequences identifiedusing one of the pertinent analytical techniques well known in the art,such as the sequences shown in Tables V-XVIII or a peptide of 8, 9, 10or 11 amino acids specified by an HLA Class I motif (e.g., Table IV (A))and/or a peptide of at least 9 amino acids that comprises an HLA ClassII motif (e.g., Table IV (B)). As is appreciated in the art, the HLAClass I binding grove is essentially closed ended so that peptides ofonly a particular size range can fit into the groove and be bound,generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. Incontrast, the HLA Class II binding groove is essentially open ended;therefore a of about 9 or more amino acids can be bound by an HLA ClassII molecule. Due to the binding groove differences between HLA Class Iand II, HLA Class I motifs are length specific, i.e., position two of aClass I motif is the second amino acid in an amino to carboxyl directionof the peptide. The amino acid positions in a Class II motif arerelative only to each other, not the overall peptide, i.e., additionalamino acids can be attached to the amino and/or carboxyl termini of amotif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long,or longer than 25 amino acids.

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. the 125P5C8 protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 125P5C8 in a host, by contacting the host with asufficient amount of at least one 125P5C8 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 125P5C8 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 125P5C8-related protein or aman-made multiepitopic peptide comprising: administering 125P5C8immunogen (e.g. the 125P5C8 protein or a peptide fragment thereof, an125P5C8 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 125P5C8 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes an 125P5C8 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). The DNA can bedissociated from an infectious agent. Optionally a genetic vaccinefacilitator such as anionic lipids; saponins; lectins; estrogeniccompounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea isalso administered.

Thus, viral gene delivery systems are used to deliver a 125P5C8-relatednucleic acid molecule. Various viral gene delivery systems that can beused in the practice of the invention include, but are not limited to,vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus,adeno-associated virus, lentivirus, and sindbis virus (Restifo, 1996,Curr. Opin. Immunol. 8:658-663). Non-viral delivery systems can also beemployed by introducing naked DNA encoding a 125P5C8-related proteininto the patient (e.g., intramuscularly or intradermally) to induce ananti-tumor response. In one embodiment, the full-length human 125P5C8cDNA is employed. In another embodiment, 125P5C8 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells to present 125P5C8 antigen to a patient'simmune system. Dendritic cells express MHC class I and II molecules, B7co-stimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al.,1996, Prostate 29:371-380). Thus, dendritic cells can be used to present125P5C8 peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed with125P5C8 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 125P5C8 protein. Yet another embodiment involves engineeringthe overexpression of the 125P5C8 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 125P5C8 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

Anti-idiotypic anti-125P5C8 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 125P5C8-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-125P5C8 antibodiesthat mimic an epitope on a 125P5C8-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

XI.) Inhibition of 125P5C8 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 125P5C8 to its binding partner or its association withother protein(s) as well as methods for inhibiting 125P5C8 function.

XI.A.) Inhibition of 125P5C8 With Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 125P5C8 are introduced into 125P5C8expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-125P5C8 antibody is expressed intracellularly,binds to 125P5C8 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol.Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to precisely target the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 125P5C8 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 125P5C8 intrabodies in orderto achieve the desired targeting. Such 125P5C8 intrabodies are designedto bind specifically to a particular 125P5C8 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to the 125P5C8protein are used to prevent 125P5C8 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 125P5C8 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XI.B.) Inhibition of 125P5C8 with Recombinant Proteins

In another approach, recombinant molecules bind to 125P5C8 and therebyinhibit 125P5C8 function. For example, these recombinant moleculesprevent or inhibit 125P5C8 from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 125P5C8specific antibody molecule. In a particular embodiment, the 125P5C8binding domain of a 125P5C8 binding partner is engineered into a dimericfusion protein, whereby the fusion protein comprises two 125P5C8 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgGl. Such IgG portion can contain, for example, the C^(H)2 and C_(H)3domains and the hinge region, but not the C_(H)1 domain. Such dimericfusion proteins are administered in soluble form to patients sufferingfrom a cancer associated with the expression of 125P5C8, whereby thedimeric fusion protein specifically binds to 125P5C8 and blocks 125P5C8interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

XI.C.) Inhibition of 125P5C8 Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 125P5C8 gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 125P5C8 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 125P5C8gene comprises contacting the 125P5C8 gene with a 125P5C8 antisensepolynucleotide. In another approach, a method of inhibiting 125P5C8 mRNAtranslation comprises contacting the 125P5C8 mRNA with an antisensepolynucleotide. In another approach, a 125P5C8 specific ribozyme is usedto cleave the 125P5C8 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the 125P5C8 gene, such as the 125P5C8 promoterand/or enhancer elements. Similarly, proteins capable of inhibiting a125P5C8 gene transcription factor are used to inhibit 125P5C8 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 125P5C8 by interferingwith 125P5C8 transcriptional activation are also useful to treat cancersexpressing 125P5C8. Similarly, factors that interfere with 125P5C8processing are useful to treat cancers that express 125P5C8. Cancertreatment methods utilizing such factors are also within the scope ofthe invention.

XI.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing 125P5C8(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 125P5C8 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 125P5C8 antisensepolynucleotides, ribozymes, factors capable of interfering with 125P5C8transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 125P5C8 to a bindingpartner, etc.

In vivo, the effect of a 125P5C8 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628, Sawyers etal., published Apr. 23, 1998, describes various xenograft models ofhuman prostate cancer capable of recapitulating the development ofprimary tumors, micrometastasis, and the formation of osteoblasticmetastases characteristic of late stage disease. Efficacy can bepredicted using assays that measure inhibition of tumor formation, tumorregression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XII.) Kits

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a 125P5C8-related protein or a 125P5C8 geneor message, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequence of FIG. 2 or analogs thereof, or a nucleic acid molecule thatencodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

A label can be present on the container to indicate that the compositionis used for a specific therapy or non-therapeutic application, and canalso indicate directions for either in vivo or in vitro use, such asthose described above. Directions and or other information can also beincluded on an insert which is included with the kit.

[***] has been deposited under the requirements of the Budapest Treatyon [***] with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209 USA, and has been identifiedas ATCC Accession No. [***].

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 125P5C8 Gene

The SSH cDNA fragment 125P5C8 (FIG. 1) was derived from a subtractionutilizing the xenografts LAPC9AD (14 days after castration) minusLAPC9AD (non-castrated mouse). The full-length cDNA clone125P5C8-Pro-pCR2.1 (FIG. 2) was identified by assembling EST fragmentshomologous to 125P5C8 into a large contiguous sequence with an ORF andamplifying the ORF by PCR using prostate first strand cDNA.

The cDNA clone 125P5C8-Pro-pCR2.1 encodes a 699 amino acid ORF with 10transmembrane domains predicted at the cell surface (PSORT). The 125P5C8protein is similar to a GenBank protein AK025164 with one amino aciddifference (FIG. 4A). This amino acid difference at amino acid position689 may be significant since it is located in the long extracellularC-terminal region that may be involved in ligand binding, may affect thestability of the protein, or may be involved in the binding of the125P5C8 protein to itself or other proteins. Protein AK025164 is novel,it was isolated from normal colon library, and has not been associatedwith any cancers. In addition, 125P5C8 is homologous to several yeastproteins, one of which is predicted to be localized to the cell surfaceand involved in sensitivity to certain drugs (FIG. 4B).

Materials and Methods LAPC Xenografts and Human Tissues:

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3: 402-408; Craftet al., 1999, Cancer Res. 59: 5030-5036). Androgen dependent andindependent LAPC-4 xenografts LAPC-4 AD and AI, respectively) and LAPC-9AD and AI xenografts were grown in male SCID mice and were passaged assmall tissue chunks in recipient males. LAPC-4 and -9 AI xenografts werederived from LAPC-4 or -9 AD tumors, respectively. To generate the AIxenografts, male mice bearing AD tumors were castrated and maintainedfor 2-3 months. After the tumors re-grew, the tumors were harvested andpassaged in castrated males or in female SCID mice.

Cell Lines:

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 5% fetal calf serum.

RNA Isolation:

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/ g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): (SEQ ID NO: 7) 5′TTTTGATCAAGCTT₃₀3′Adaptor 1: (SEQ ID NO: 8) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′(SEQ ID NO: 9) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 10)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 11)3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO: 12) 5′CTAATACGACTCACTATAGGGC3′Nested primer (NP)1: (SEQ ID NO: 13) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nestedprimer (NP)2: (SEQ ID NO: 14) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from two LAPC-9 AD xenografts.Specifically, to isolate genes that are involved in the progression ofandrogen dependent (AD) prostate cancer to androgen independent (AI)cancer, an experiment was conducted with the LAPC-9 AD xenograft in maleSCID mice. Mice that harbored LAPC-9 AD xenografts were castrated whenthe tumors reached a size of 1 cm in diameter. The tumors regressed insize and temporarily stopped producing the androgen dependent proteinPSA. Seven to fourteen days post-castration, PSA levels were detectableagain in the blood of the mice. Eventually the tumors develop an AIphenotype and start growing again in the castrated males. Tumors wereharvested at different time points after castration to identify genesthat are turned on or off during the transition to androgenindependence.

The gene 125P5C8 was derived from an LAPC-9 AD tumor (14 dayspost-castration) minus an LAPC-9 AD tumor (grown in intact male mouse)subtraction. The SSH DNA sequence of 278 bp (FIG. 1) was identified.

The cDNA derived from an LAPC-9 AD tumor (14 days post-castration) wasused as the source of the “tester” cDNA, while the cDNA from the LAPC-9AD tumor (grown in intact male mouse) was used as the source of the“driver” cDNA. Double stranded cDNAs corresponding to tester and drivercDNAs were synthesized from 2 μg of poly(A)⁺ RNA isolated from therelevant xenograft tissue, as described above, using CLONTECH'sPCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN asprimer. First- and second-strand synthesis were carried out as describedin the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1,Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1)and ethanol precipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digestedcDNA from the relevant xenograft source (see above) with a mix ofdigested cDNAs derived from the human cell lines HeLa, 293, A431,Colo205, and mouse liver.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant xenograft source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10×reaction buffer (CLONTECH) and0.5 μl 50×Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:15) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 16) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1X Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: Initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 b.p.β-actin bands from multiple tissues were compared by visual inspection.Dilution factors for the first strand cDNAs were calculated to result inequal β-actin band intensities in all tissues after 22 cycles of PCR.Three rounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 125P5C8 gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification. Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities.

In a typical RT-PCR Expression analysis shown in FIG. 5. RT-PCRexpression analysis was performed on first strand cDNAs generated usingpools of tissues from multiple samples. The cDNAs were subsequentlynormalized using beta-actin PCR. Expression of 125P5C8 was observed inprostate cancer xenografts, normal prostate tissue pools, prostatecancer tissue pools, colon cancer tissue pools, kidney cancer tissuepools, and bladder cancer tissue pools.

Example 2 Full Length Cloning of 125P5C8 and Homology Comparison toKnown Sequences

To isolate genes that are involved in the progression of androgendependent (AD) prostate cancer to androgen independent (AI) cancer, weconducted an experiment with the LAPC-4 AD xenograft in male SCID mice.Mice that harbored LAPC-9 AD xenografts were castrated when the tumorsreached a size of 1 cm in diameter. The tumors regressed in size andtemporarily stopped producing the androgen dependent protein PSA. Sevento fourteen days post-castration, PSA levels were detectable again inthe blood of the mice. Eventually the tumors develop an Al phenotype andstart growing again in the castrated males. Tumors were harvested atdifferent time points after castration to identify genes that are turnedon or off during the transition to androgen independence.

The gene 125P5C8 was derived from an LAPC-9 AD (14 days post-castration)minus LAPC-9 AD (no castration) subtraction. The SSH DNA sequence of 287bp (FIG. 1) was designated 125P5C8. The full-length cDNA clone125P5C8-Pro-pCR2.1 (FIG. 2) was identified by assembling EST fragmentshomologous to 125P5C8 into a large contiguous sequence with an ORF andamplifying the ORF by PCR using prostate first strand cDNA. The cDNAclone 125P5C8-Pro-pCR2.1 encodes a 699 amino acid ORF with 10transmembrane domains predicted at the cell surface based on PSORTanalysis (http://psort.nibb.ac.jp:8800/form.html).

The 125P5C8 protein is expected to be a cell surface protein based ontopology algorithms. This can be confirmed by IHC, immunofluorescence,flow cytometry and cell fractionation techniques, using engineered celllines, as well as non-engineered cell lines and primary tissues thatexpress 125P5C8. When 125P5C8 is expressed at the cell surface, it isused as a target for diagnostic, preventative and therapeutic purposes.

The 125P5C8 protein is similar to GenBank protein AK025164 with oneamino acid difference (FIG. 4A). This amino acid difference at aminoacid position 689 may be significant since it is located in the longextracellular C-terminal region that may be involved in signaltransduction, ligand binding, may affect the stability of the protein,or may be involved in the binding of the 125P5C8 protein to itself orother proteins. In addition, 125P5C8 is homologous to several yeastproteins, one of which is predicted to be localized to the cell surfaceand be involved in sensitivity to certain drugs (FIG. 4B).

At the protein level, 125P5C8 shows 33% identity and 49% homology toYCR017 (SPAC589), a 5-transmembrane containing yeast protein proposed toplay a role in drug sensitivity. In addition, 125P5C8 has 23% identityand 20% homology to an ABC transporter (E81015) containing 13transmembrane domains. Most of the homology to the ABC transporter waslocated between amino acids 80-330 of 125P5C8, and overlaps with one ofthe transporter motifs of the ABC protein. Based on protein motifspresent in 125P5C8 as well as homology analysis, 125P5C8 can function as(1) a protein transporter or drug resistance gene, (2) an ion symporteror (3) ion channel.

The 125P5C8 cDNA was deposited on [***] 2001 with the American TypeCulture Collection (ATCC; Manassas, Va.) as plasmid [***], and has beenassigned Accession No. PTA-[***].

Example 3 Chromosomal Mapping of the 125P5C8 Gene

The chromosomal localization of 125P5C8 was determined using the NCBIHuman Genome web site(http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).The mapping program placed 125P5C8 on chromosome 6q23, between D6S1040and D6S457, a genomic region found to be rearranged in certain cancers.

Example 4 Expression Analysis of 125P5C8 in Normal Tissues, Cancer CellLines and Patient Samples

Expression analysis by RT-PCR demonstrated that normal tissue expressionof 125P5C8 is restricted predominantly to prostate and, to lower extent,it is detected in a pool of kidney, liver and lung (FIG. 5). Analysis ofhuman cancer patient RNA pools showed expression in prostate, bladderkidney, as well as colon cancers (FIG. 5).

Extensive Northern blot analysis of 125P5C8 in 16 human normal tissuesconfirms the expression observed by RT-PCR (FIGS. 6A and 6B). A 3 kbtranscript is detected in normal prostate and to lower extent in colonand kidney. 125P5C8 expression was also shown in prostate cancerxenografts (FIG. 6C). Expression is highest in LAPC4AI and LAPC9AI whencompared to the androgen dependent counterparts suggesting a role inacquiring androgen independent tumor growth.

Northern blot analysis shows that 125P5C8 is expressed in prostate tumortissues derived from prostate cancer patients (FIG. 7). One of fivetumor-normal prostate pairs shows over-expression in tumor. Theexpression of 125P5C8 in kidney cancer is down-regulated when comparedto the normal and adjacent kidney tissues (FIG. 8).

Biological Relevance

The expression pattern of 125P5C8 is prostate-restricted. In normaltissues 125P5C8 is only expressed in prostate and at lower levels inkidney and colon. High expression is also seen in prostate cancerxenografts and in prostate cancer patient samples. Accordingly, theexpression pattern of 125P5C8 indicates its utility in prostate cancer.

Therapeutic applications for 125P5C8 include use as a small moleculetherapy and/or a vaccine (T cell or antibody) target. Diagnosticapplications for 125P5C8 include use as a diagnostic marker for localand/or metastasized disease. 125P5C8 is expressed in the LAPC-4 andLAPC-9 xenografts that are derived from lymph node and bone metastasisof prostate cancer, respectively. The restricted expression of 125P5C8in normal tissues makes it useful as a tumor target for diagnosis andtherapy. 125P5C8 expression analysis provides information useful forpredicting susceptibility to advanced stage disease, rate ofprogression, and/or tumor aggressiveness. 125P5C8 expression status inpatient samples, tissue arrays and/or cell lines may be analyzed by: (i)immunohistochemical analysis; (ii) in situ hybridization; (iii) RT-PCRanalysis on laser capture micro-dissected samples; (iv) Western blotanalysis; and (v) Northern analysis.

Example 5 Generation of 125P5C8 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Forexample, 125P5C8, recombinant bacterial fusion proteins or peptidesencoding various regions of the 125P5C8 sequence are used to immunizeNew Zealand White rabbits. Typically a peptide can be designed from acoding region of 125P5C8. The peptide can be conjugated to keyholelimpet hemocyanin (KLH) and used to immunize a rabbit. Alternatively theimmunizing agent may include all or portions of the 125P5C8 protein,analogs or fusion proteins thereof. For example, the 125P5C8 amino acidsequence can be fused to any one of a variety of fusion protein partnersthat are well known in the art, such as maltose binding protein, LacZ,thioredoxin or an immunoglobulin constant region (see e.g. CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubulet al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L.,Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566). Otherrecombinant bacterial proteins include glutathione-S-transferase (GST),and HIS tagged fusion proteins of 125P5C8 that are purified from inducedbacteria using the appropriate affinity matrix.

It may be useful to conjugate the immunizing agent to a protein known tobe immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 5-50 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant. Rabbits are theninjected subcutaneously every two weeks with up to 200 μg, typically5-50 μg, of immunogen in incomplete Freund's adjuvant. Test bleeds aretaken approximately 7-10 days following each immunization and used tomonitor the titer of the antiserum by ELISA.

To test serum, such as rabbit serum, for reactivity with 125P5C8proteins, the full-length 125P5C8 cDNA can be cloned into an expressionvector such as one that provides a 6His tag at the carboxyl-terminus(pCDNA 3.1 myc-his, Invitrogen). After transfection of the constructsinto 293T cells, cell lysates can be probed with anti-His antibody(Santa Cruz Biotechnologies, Santa Cruz, Calif.) and the anti-125P5C8serum using Western blotting. Alternatively specificity of the antiserumis tested by Western blot and immunoprecipitation analyses using lysatesof cells that express 125P5C8. Serum from rabbits immunized with GST orMBP fusion proteins is first semi-purified by removal of anti-GST oranti-MBP antibodies by passage over GST and MBP protein columnsrespectively. Sera from His-tagged protein and peptide immunized rabbitsas well as depleted GST and MBP protein sera are purified by passageover an affinity column composed of the respective immunogen covalentlycoupled to Affigel matrix (BioRad).

Example 6 Generation of 125P5C8 Monoclonal Antibodies (MAbs)

In one embodiment, therapeutic MAbs to 125P5C8 will include those thatreact with extracellular epitopes of 125P5C8. Immunogens for generationof such MAbs are designed to encode or contain extracellular regions ofthe 125P5C8 protein predicted from protein topology algorithms. Theseimmunogens include peptides, recombinant bacterial proteins, andmammalian expressed Tag5 proteins and human and murine IgG FC fusionproteins. The membrane topology of 125P5C8 is that of a 10 transmembraneprotein (Table XIX) with the amino-terminus embedded in the membrane andthe long carboxy terminus extracellular (Sosui topology prediction).Thus, extracellular regions useful to include in immunogen design arethe carboxy terminus encoding amino acids 412-699 andinter-transmembrane sequences encoding amino acids 65-93, amino acids143-188, amino acids 261-268, and amino acids 341-349. Alternatively, ifthe carboxy terminus is intracellular, extracellular regions useful toinclude in antigen design are amino acids 24-41, amino acids 117-119,amino acids 212-237, amino acids 292-317, and amino acids 369-389. Togenerate MAbs to 125P5C8, mice are first immunized intraperitoneally(IP) with up to 200 μg, typically 10-50 μg, of protein immunogen mixedin complete Freund's adjuvant. Mice are then subsequently immunized IPevery 2-4 weeks with up to 200 μg , typically 10-50 μg, of antigen mixedin Freund's incomplete adjuvant. Alternatively, Ribi adjuvant is usedimmunizations. In addition, a DNA-based immunization protocol isemployed in which a mammalian expression vector encoding 125P5C8sequence is used to immunize mice by direct injection of the plasmidDNA. For example, pCDNA 3.1 encoding either the full length 125P5C8 cDNAor extracellular coding regions of 125P5C8 fused to the coding sequenceof murine or human IgG are used. This protocol is used alone or incombination with protein immunogens. Test bleeds are taken 7-10 daysfollowing immunization to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, and immunoprecipitation analyses,fusion and hybridoma generation is then carried with establishedprocedures well known in the art (Harlow and Lane, 1988).

In one embodiment for generating 125P5C8 monoclonal antibodies, aglutathione-S-transferase (GST) fusion protein encompassing thecarboxy-terminal domain of 125P5C8 (amino acids 412-699) is expressed,purified, and used as immunogen. Balb C mice are initially immunizedintraperitoneally with 25 μg of the GST-125P5C8 fusion protein mixed incomplete Freund's adjuvant. Mice are subsequently immunized every twoweeks with 25 μg of GST-125P5C8 protein mixed in Freund's incompleteadjuvant for a total of three immunizations. To determine titer of serumfrom immunized mice, ELISA is carried out using a 125P5C8-specificcleavage fragment of the immunogen in which GST is removed by sitespecific proteolysis. Reactivity and specificity of serum to full length125P5C8 protein is monitored by Western blotting and flow cytometryusing 293T cells transfected with an expression vector encoding the125P5C8 cDNA (Example 7). Mice showing the strongest reactivity arerested for three weeks and given a final injection of 125P5C8 cleavagefragment in PBS and then sacrificed four days later. The spleens of thesacrificed mice are then harvested and fused to SPO/2 myeloma cellsusing standard procedures (Harlow and Lane, 1988). Supernatants fromgrowth wells following HAT selection are screened by ELISA, Westernblot, and flow cytometry to identify 125P5C8 specific antibody-producingclones.

The binding affinity of a 125P5C8 monoclonal antibody is determinedusing standard technologies. Affinity measurements quantify the strengthof antibody to epitope binding and can be used to help define which125P5C8 monoclonal antibodies are preferred for diagnostic ortherapeutic use. The BIAcore system (Uppsala, Sweden) is a preferredmethod for determining binding affinity. The BlAcore system uses surfaceplasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Mortonand Myszka, 1998, Methods in Enzymology 295: 268) to monitorbiomolecular interactions in real time. BlAcore analysis convenientlygenerates association rate constants, dissociation rate constants,equilibrium dissociation constants, and affinity constants.

Example 7 Production of Recombinant 125P5C8 in Bacterial and MammalianSystems Bacterial Constructs

pGEX Constructs

To express 125P5C8 in bacterial cells, portions of 125P5C8 are fused tothe Glutathione S-transferase (GST) gene by cloning into pGEX-6P-1(Amersham Pharmacia Biotech, NJ). The constructs were made in order togenerate recombinant 125P5C8 protein sequences with GST fused at theN-terminus and a six histidine epitope at the C-terminus. The sixhistidine epitope tag is generated by adding the histidine codons to thecloning primer at the 3′ end of the open reading frame (ORF). APreScission™ recognition site permits cleavage of the GST tag from125P5C8-related protein. The ampicillin resistance gene and pBR322origin permits selection and maintenance of the plasmid in E. coli. Forexample, cDNA encoding the following fragments of 125P5C8 protein werecloned into pGEX-6P-1: amino acids 1 to 141; amino acids 142 to 288;amino acids 142 to 188, amino acids 188 to 410; and amino acids 411 to699, or any 8, 9, 10, 11, 12, 13, 14 or 15 contiguous amino acids from125P5C8 or an analog thereof.

pMAL Constructs

To express 125P5C8 in bacterial cells, all or part of the 125P5C8nucleic acid sequence are fused to the maltose-binding protein (MBP)gene by cloning into pMAL-c2X and pMAL-p2X (New England Biolabs, MA).The constructs are made to generate recombinant 125P5C8 proteinsequences with MBP fused at the N-terminus and a six histidine epitopeat the C-terminus. The six histidine epitope tag is generated by addingthe histidine codons to the 3′ cloning primer. A Factor Xa recognitionsite permits cleavage of the GST tag from 125P5C8. The pMAL-c2X andpMAL-p2X vectors are optimized to express the recombinant protein in thecytoplasm or periplasm respectively. Periplasm expression enhancesfolding of proteins with disulfide bonds. For example, cDNA encoding thefollowing fragments of 125P5C8 protein are cloned into pMAL-c2X andpMAL-p2X: amino acids 1 to 141; amino acids 142 to 288; amino acids 142to 188, amino acids 188 to 410; and amino acids 411 to 699, or any 8, 9,10, 11, 12, 13, 14 or 15 contiguous amino acids from 125P5C8 or ananalog thereof.

pCRII

To generate 125P5C8 sense and anti-sense riboprobes for RNA in situinvestigations, a pCRII construct (Invitrogen, Carlsbad Calif.) isgenerated using cDNA sequence encoding amino acids 1 to 141, and 142 to288. The pCRII vector has Sp6 and T7 promoters flanking the insert todrive the production of 125P5C8 RNA riboprobes which are used in RNA insitu hybridization experiments.

Mammalian Constructs

To express recombinant 125P5C8, the full or partial length 125P5C8 cDNAcan be cloned into any one of a variety of expression vectors known inthe art. The constructs can be transfected into any one of a widevariety of mammalian cells such as 293T cells. Transfected 293T celllysates can be probed with the anti-125P5C8 polyclonal serum, describedin Example 5 above, in a Western blot.

The 125P5C8 genes can also be subcloned into the retroviral expressionvector pSRαMSVtkneo and used to establish 125P5C8-expressing cell linesas follows: The 125P5C8 coding sequence (from translation initiation ATGand Kozak translation start consensus sequence to the terminationcodons) is amplified by PCR using ds cDNA template from 125P5C8 cDNA.The PCR product is subcloned into pSRαMSVtkneo vector and transformedinto DH5α competent cells. Colonies are picked to screen for clones withunique internal restriction sites on the cDNA. The positive clone isconfirmed by sequencing of the cDNA insert. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, NIH 3T3, TsuPrl, 293 or rat-1 cells.

Additional illustrative mammalian and bacterial systems are discussedbelow.

pcDNA4/HisMax-TOPO Constructs

To express 125P5C8 in mammalian cells, the 125P5C8 ORF is cloned intopcDNA4/HisMax-TOPO Version A (cat# K864-20, Invitrogen, Carlsbad,Calif.). Protein expression is driven from the cytomegalovirus (CMV)promoter and the SP163 translational enhancer. The recombinant proteinhas Xpress™ and six histidine epitopes fused to the N-terminus to aid indetection and purification of the recombinant protein. ThepcDNA4/HisMax-TOPO vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColElorigin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs

To express 125P5C8 in mammalian cells, the ORF with consensus Kozaktranslation initiation site was cloned into pcDNA3.1/MycHis_Version A(Invitrogen, Carlsbad, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant protein has the mycepitope and six histidines fused to the C-terminus to aid in detectionand purification of the recombinant protein. The pcDNA3.1/MycHis vectoralso contains the bovine growth hormone (BGH) polyadenylation signal andtranscription termination sequence to enhance mRNA stability, along withthe SV40 origin for episomal replication and simple vector rescue incell lines expressing the large T antigen. The Neomycin resistance genecan be used, as it allows for selection of mammalian cells expressingthe protein and the ampicillin resistance gene and ColE1 origin permitsselection and maintenance of the plasmid in E. coli.

pcDNA3.1/V5His-TOPO Constructs

To express 125P5C8 in mammalian cells, the cDNA encoding the 125P5C8 ORFand Kozak consensus translation initiation sequence is cloned intopcDNA4/V5His-TOPO (cat# K4800-01, Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter. Therecombinant protein has V5™ and six histidine epitopes fused at theC-terminus to aid in detection and purification of the recombinantprotein. The pcDNA4/V5His-TOPO vector also contains the bovine growthhormone (BGH) polyadenylation signal and transcription terminationsequence to enhance mRNA stability along with the SV40 origin forepisomal replication and simple vector rescue in cell lines expressingthe large T antigen. The Zeocin resistance gene allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli.

pcDNA3.1CT-GFP-TOPO Construct

To express 125P5C8 in mammalian cells and to allow detection of therecombinant protein using fluorescence, the ORF with consensus Kozaktranslation initiation site is cloned into pcDNA3.1CT-GFP-TOPO(Invitrogen, CA). Protein expression is driven from the cytomegalovirus(CMV) promoter. The recombinant protein has the Green FluorescentProtein (GFP) fused to the C-terminus facilitating non-invasive, in vivodetection and cell biology studies. The pcDNA3.1/MycHis vector alsocontains the bovine growth hormone (BGH) polyadenylation signal andtranscription termination sequence to enhance mRNA stability along withthe SV40 origin for episomal replication and simple vector rescue incell lines expressing the large T antigen. The Neomycin resistance geneallows for selection of mammalian cells that express the protein, andthe ampicillin resistance gene and ColE1 origin permits selection andmaintenance of the plasmid in E. coli. An additional construct with aN-terminal GFP fusion is made in pcDNA3.1NT-GFP-TOPO spanning the entirelength of the 125P5C8 protein.

pAPtag Constructs

The cDNA encoding 125P5C8 amino acids 142-188 and 411-699 are clonedinto pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This constructgenerates an alkaline phosphatase fusion at the C-terminus of the125P5C8 protein while fusing the IgGK signal sequence to N-terminus. Theresulting recombinant 125P5C8 protein is optimized for secretion intothe media of transfected mammalian cells and can be used to identifyproteins such as ligands or receptors that interact with the 125P5C8protein. Protein expression is driven from the CMV promoter and therecombinant protein also contains myc and six histidines fused to theC-terminus of alkaline phosphatase to aid in detection and purificationof the recombinant protein. The Zeosin resistance gene allows forselection of mammalian cells expressing the protein and the ampicillinresistance gene permits selection of the plasmid in E. coli.

ptag5 Constructs

The cDNA encoding for 125P5C8 amino acids 142-188 and 411-699 are clonedinto pTag-5. This vector is similar to pAPtag but without the alkalinephosphatase fusion. This construct generates an immunoglobulin G1 Fcfusion at the C-terminus of the 125P5C8 protein while fusing the IgGKsignal sequence to the N-terminus. The resulting recombinant 125P5C8protein is optimized for secretion into the media of transfectedmammalian cells, and can be used to identify proteins such as ligands orreceptors that interact with the 125P5C8 protein. Protein expression isdriven from the CMV promoter and the recombinant protein also containsmyc and six histidines fused to the C-terminus to aid in detection andpurification of the recombinant protein. The Zeocin resistance geneallows for selection of mammalian cells expressing the protein, and theampicillin resistance gene permits selection of the plasmid in E. coli.

psecFc Constructs

The cDNA encoding for 125P5C8 amino acids 142-188 and 411-699 are clonedinto psecFc. The psecFc vector was assembled by cloning immunoglobulinG1 Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California).This construct generates an immunoglobulin G1 Fc fusion at theC-terminus of the 125P5C8 protein, while fusing the IgGK signal sequenceto N-terminus. The resulting recombinant 125P5C8 protein is optimizedfor secretion into the media of transfected mammalian cells, and can beused to identify proteins such as ligands or receptors that interactwith the 125P5C8 protein. Protein expression is driven from the CMVpromoter. The Zeocin resistance gene allows for selection of mammaliancells that express the protein, and the ampicillin resistance genepermits selection of the plasmid in E. coli.

pSRαConstructs

To generate mammalian cell lines that express 125P5C8 constitutively,the ORF is cloned into pSRα constructs. Amphotropic and ecotropicretroviruses are generated by transfection of pSRα constructs into the293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid((φ˜) in the 293 cells, respectively. The retrovirus can be used toinfect a variety of mammalian cell lines, resulting in the integrationof the cloned gene, 125P5C8, into the host cell-lines. Proteinexpression is driven from a long terminal repeat (LTR). The Neomycinresistance gene allows for selection of mammalian cells that express theprotein, and the ampicillin resistance gene and ColE1 origin permitselection and maintenance of the plasmid in E. coli.

An additional pSRα construct was made that fused the FLAG tag to theC-terminus to allow detection using anti-FLAG antibodies. The FLAGsequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 6) were addedto cloning primer at the 3′ end of the ORF.

Additional pSRα constructs are made to produce both N-terminal andC-terminal GFP and myc/6 HIS fusion proteins of the full-length 125P5C8protein.

Example 8 Production of Recombinant 125P5C8 in a Baculovirus System

To generate a recombinant 125P5C8 protein in a baculovirus expressionsystem, cDNA sequence encoding the 125P5C8 protein is cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus Specifically, pBlueBac-125P5C8 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 125P5C8 protein is then generated by infection of HighFiveinsect cells (Invitrogen) with the purified baculovirus. Recombinant125P5C8 protein can be detected using anti-125P5C8 antibody. 125P5C8protein can be purified and used in various cell-based assays or asimmunogen to generate polyclonal and monoclonal antibodies specific for125P5C8.

Example 9 Identification of Potential Signal Transduction Pathways

Transporters have been reported to interact with a variety of signalingmolecules and regulate signaling pathways (J Neurochem. 2001;76:217-223). Using immunoprecipitation and Western blotting techniques,we can identify proteins that associate with 125P5C8 and mediatesignaling events. Several pathways known to play a role in cancerbiology can be regulated by 125P5C8, including phospholipid pathwayssuch as PI3K, AKT, etc, as well as mitogenic/survival cascades such asERK, p38, etc (Cell Growth Differ. 2000,11:279; J Biol Chem. 1999,274:801; Oncogene. 2000, 19:3003.). Using Western blotting techniques,we can evaluate the role that 125P5C8 plays in the regulation of thesepathways. Cells lacking 125P5C8 and cells expressing 125P5C8 are eitherleft untreated or stimulated with cytokines, androgen and anti-integrinAb. Cell lysates are analyzed using anti-phosphos-specific antibodies(Cell Signaling, Santa Cruz Biotechnology) in order to detectphosphorylation and regulation of ERK, p38, AKT, PI3K, PLC and othersignaling molecules. When 125P5C8 plays a role in the regulation ofsignaling pathways, 125P5C8 is used as a target for diagnostic,preventative and therapeutic purposes.

To determine whether 125P5C8 directly or indirectly activates knownsignal transduction pathways in cells, luciferase (luc) basedtranscriptional reporter assays are carried out in cells expressing125P5C8. These transcriptional reporters contain consensus-binding sitesfor known transcription factors that lie downstream ofwell-characterized signal transduction pathways. The reporters andexamples of these associated transcription factors, signal transductionpathways, and activation stimuli are listed below.

-   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress-   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation-   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress-   4. ARE-luc, androgen receptor; steroids/MAPK;    growth/differentiation/apoptosis-   5. p53-luc, p53; SAPK; growth/differentiation/apoptosis-   6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

125P5C8-mediated effects can be assayed in cells showing mRNAexpression. Luciferase reporter plasmids can be introduced bylipid-mediated transfection (TFX-50, Promega). Luciferase activity, anindicator of relative transcriptional activity, is measured byincubation of cell extracts with luciferin substrate and luminescence ofthe reaction is monitored in a luminometer.

Signaling pathways activated by 125P5C8 are mapped and used for theidentification and validation of therapeutic targets in the 125P5C8pathway. When 125P5C8 is involved in cell signaling, it is used as atarget for diagnostic, preventative and therapeutic purposes.

Example 10 Involvement of 125P5C8 in Tumor Progression

125P5C8 can contribute to the growth of cancer cells. The role of125P5C8 in tumor growth is investigated in prostate, colon and kidneycell lines as well as NIH 3T3 cells engineered to stably express125P5C8. Parental 125P5C8 negative cells and 125P5C8-expressing cellsare evaluated for cell growth using a well-documented proliferationassay (Fraser S P, Grimes J A, Djamgoz M B. Prostate. 2000;44:61,Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288).

To determine the role of 125P5C8 in the transformation process, weinvestigate its effect in colony forming assays. Parental NIH3T3 cellslacking 125P5C8 are compared to NHI-3T3-125P5C8 cells in a soft agarassay under stringent and more permissive conditions (Song Z. et al.Cancer Res. 2000;60:6730).

To determine the role of 125P5C8 in invasion and metastasis of cancercells, we use a well-established Transwell Insert System assay (BectonDickinson) (Cancer Res. 1999; 59:6010). Cells lacking 125P5C8 and cellsexpressing 125P5C8 are loaded with the fluorescent dye, calcein, andplated in the top well of the Transwell insert. Invasion is determinedby fluorescence of cells in the lower chamber relative to thefluorescence of the entire cell population.

125P5C8 can also play a role in cell cycle and apoptosis. PC3-125P5C8cells are compared to 125P5C8-negative PC3 for differences in cell cycleregulation using a well-established BrdU assay (Abdel-Malek Z A. J CellPhysiol. 1988, 136:247). In short, cells are grown under both optimal(full serum) and limiting (low serum) conditions are labeled with BrdUfor 1 hour and stained with anti-BrdU Ab and propidium iodide. Cells areanalyzed for entry into the G1, S, and G2M phases of the cell cycle.Alternatively, the effect of stress on apoptosis is evaluated in125P5C8-negative cells and 125P5C8-expressing cells, including normaland tumor prostate, colon and lung cells. Engineered and parental cellstreated with various chemotherapeutic agents and protein synthesisinhibitors are stained with annexin V-FITC. Cell death is measured byFACS analysis.

The effect of 125P5C8 on stress- and chemotherapeutic-induced cell deathcan be evaluated by FACS. 125P5C8-negative cells and 125P5C8-expressingcells are treated with various chemotherapeutic agents, such asetoposide, flutamide, etc, and protein synthesis inhibitors, such ascycloheximide. The cells are stained with annexin V-FITC and cell deathis measured.

Furthermore, 125P5C8-expressing cells, such as normal and tumorprostate, colon and lung cells, are compared to 125P5C8-negative cellsfor differences in cell cycle regulation using a well-established BrdUassay (Abdel-Malek Z A. J Cell Physiol. 1988, 136:247). In short, cellsgrown under both optimal (full serum) and limiting (low serum)conditions are labeled with BrdU for 1 hour and stained with anti-BrdUAb and propidium iodide. Cells are analyzed for entry into the G1, S,and G2M phases of the cell cycle.

When 125P5C8 plays a role in cell growth, transformation, invasion orapoptosis, it is used as a target for diagnostic, preventative andtherapeutic purposes.

Example 11 Western Analysis of 125P5C8 Expression in SubcellularFractions

The cellular location of 125P5C8 can be assessed using subcellularfractionation techniques widely used in cellular biology (Storrie B, etal. Methods Enzymol. 1990;182:203-25). Prostate or other cell lines canbe separated into nuclear, cytosolic and membrane fractions. Theexpression of 125P5C8 in the different fractions can be tested usingWestern blotting techniques.

Alternatively, to determine the subcellular localization of 125P5C8,293T cells can be transfected with an expression vector encodingHIS-tagged 125P5C8 (PCDNA 3.1 MYC/HIS, Invitrogen). The transfectedcells can be harvested and subjected to a differential subcellularfractionation protocol as previously described (Pemberton, P. A. et al,1997, J of Histochemistry and Cytochemistry, 45:1697-1706.) Thisprotocol separates the cell into fractions enriched for nuclei, heavymembranes (lysosomes, peroxisomes, and mitochondria), light membranes(plasma membrane and endoplasmic reticulum), and soluble proteins.

Example 12 Protein Transporter Function

Using a modified rhodamine retention assay (Davies J et al. Science2000, 290:2295; Leith C et al. Blood 1995, 86:2329) one can determinewhether 125P5C8 functions as a protein transporter. Cell lines, such asprostate, colon and kidney cancer and normal cells, expressing orlacking 125P5C8 are loaded with Calcein AM (Molecular Probes). Cells areexamined over time for dye transport using a fluorescent microscope orfluorometer. Quantitation is performed using a fluorometer (Hollo Z. etal. 1994. 1191:384). Information obtained from such experiments is usedin determining whether 125P5C8 extrudes chemotherapeutic drugs, such asdoxorubicin, paclitaxel, etoposide, etc, from tumor cells, therebylowering drug content and reducing tumor responsiveness to treatment.Using this technique, we are able to identify substrates for 125P5C8,and determine which drugs may be more efficacious in treating individualpatients. When 125P5C8 functions as a protein transporter, 125P5C8 isused as a target for preventative and therapeutic purposes as well asdrug sensitivity/resistance.

The function of 125P5C8 as an ion channel is determined by FACS analysisand electrophysiology (Gergely L, Cook L, Agnello V. Clin Diagn LabImmunol. 1997;4:70; Skryma R, et al. J Physiol. 2000, 527: 71). UsingFACS analysis and commercially available indicators (Molecular Probes),the ability of parental cells and cells expressing 125P5C8 to transportcalcium, sodium and potassium is compared. Prostate, colon and kidneynormal and tumor cell lines are used in these studies. For example cellsloaded with calcium responsive indicators such as Fluo4 and Fura red areincubated in the presence or absence of ions and analyzed by flowcytometry. Information derived from these experiments provides amechanism by which cancer cells are regulated. This is particularly truein the case of calcium, as calcium channel inhibitors have been reportedto induce the death of certain cancer cells, including prostate cancercell lines (Batra S, Popper L D, Hartley-Asp B. Prostate. 1991,19: 299).

The 125P5C8 protein can function as a sodium symporter. In this case,125P5C8 co-transports ions and/or proteins along with sodium. Severalmolecules have been identified to co-transport with Na+, the most commonbeing iodide. The sodium/iodide co-transporter was shown to beover-expressed in breast cancer and to play a role in iodide uptake inthyroid cancer cells (Tazebay U et al. Nat. Med. 2000, 6:871; Filetti Set al. Eur. J. Endocrinol. 1999. 141: 443). In addition, thesodium/iodide symporter has been associated with radioiodine treatmentmodality in prostate and thyroid cancer (Spitzweg C et al. Cancer Res.2000, 60:6526). In these studies ¹³¹I was (1) injected into tumor cellsin vivo experiment or (2) used to bathe tumor cells in vitro. In eithercase, accumulation of ¹³¹I induced tumor cell death. The function of125P5C8 as a co-transporter of iodide and sodium is studied using FACSanalysis techniques as well as labeled ¹³¹I. This study is critical inlight of the importance of Na+/I− transporter in therapy. When 125P5C8is a sodium symporter, it is used as a target for diagnostic,preventative and therapeutic purposes.

Using electrophysiology, uninjected oocytes and oocytes injected with125P5C8 cRNA are compared for ion channel activity. Patch/voltage clampassays are performed on oocytes in the presence or absence of selectedions, including calcium, potassium, sodium, etc. Ion channel activators(such as cAMP/GMP, forskolin, TPA, etc) and inhibitors (such ascalcicludine, conotoxin, TEA, tetrodotoxin, etc) are used to evaluatethe function of 125P5C8 as an ion channel. When 125P5C8 functions as anion channel, it is used as a target for diagnostic, preventative andtherapeutic purposes.

Example 13 Involvement of 125P5C8 in Cell-Cell Communication

Multi-transmembrane proteins have the ability to mediate intercellularcommunications. Cell expressing 125P5C8 are compared to cells lacking125P5C8 using two types of assays (J. Biol. Chem. 2000, 275:25207). Inthe first assay, cells loaded with a fluorescent dye are incubated inthe presence of unlabeled recipient cells and the cell populations areexamined under fluorescent microscopy. This qualitative assay measuresthe exchange of dye between adjacent cells. In the second assay system,donor and recipient cell populations are treated as above andquantitative measurements of the recipient cell population are performedby FACS analysis. Using these two assay systems, we can determinewhether 125P5C8 enhances or suppresses cell communications, and whethersmall molecules and/or specific antibodies modulate the function of125P5C8.

When 125P5C8 function in cell-cell communication, it is used as a targetfor diagnostic, preventative and therapeutic purposes

Example 14 Regulation of Transcription by 125P5C8

The 125P5C8 protein can play a role in transcriptional regulation ofeukaryotic genes. Regulation of gene expression can be evaluated bystudying gene expression in cells expressing or lacking 125P5C8. Forthis purpose, two types of experiments are performed. In the first setof experiments, RNA from parental and 125P5C8-expressing NIH3T3 and PC3cells, respectively, are extracted and hybridized to commerciallyavailable gene arrays (Clontech). Resting cells as well as cells treatedwith FBS or androgen are compared. Differentially expressed genes areidentified in accordance with procedures known in the art. Thedifferentially expressed genes are then mapped to biological pathways.

In the second set of experiments, specific transcriptional pathwayactivation is evaluated using commercially available (Stratagene)luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc,ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters containconsensus binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways, andrepresent a good tool to ascertain pathway activation and screen forpositive and negative modulators of pathway activation.

When 125P5C8 plays a role in gene regulation, 125P5C8 is used as atarget for diagnostic, prognostic, preventative and therapeuticpurposes.

Example 15 In Vivo Assay for 125P5C8 Tumor Growth Promotion

The effect of the 125P5C8 protein on tumor cell growth can be evaluatedin vivo by gene overexpression in tumor-bearing mice. For example, SCIDmice can be injected SQ on each flank with 1×10⁶ of either PC3, TSUPR1,or DU145 cells containing tkNeo empty vector or 125P5C8. At least twostrategies may be used: (1) Constitutive 125P5C8 expression underregulation of a promoter such as a constitutive promoter obtained fromthe genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, provided such promoters are compatible with thehost cell systems. (2) Regulated expression under control of aninducible vector system, such as ecdysone, tet, etc., can be usedprovided such promoters are compatible with the host cell systems. Tumorvolume is then monitored at the appearance of palpable tumors and isfollowed over time to determine if 125P5C8-expressing cells grow at afaster rate and whether tumors produced by 125P5C8-expressing cellsdemonstrate characteristics of altered aggressiveness (e.g. enhancedmetastasis, vascularization, reduced responsiveness to chemotherapeuticdrugs). Additionally, mice can be implanted with 1×10⁵ of the same cellsorthotopically to determine if 125P5C8 has an effect on local growth inthe prostate or on the ability of the cells to metastasize, specificallyto lungs, lymph nodes, and bone marrow. Also see Saffran et al,“Anti-PSCA mAbs inhibit tumor growth and metastasis formation andprolong the survival of mice bearing human prostate cancer xenografts”PNAS 10:1073-1078 or www.pnas.org/cgi/doi/10.1073/pnas.051624698.

The assay is also useful to determine the 125P5C8 inhibitory effect ofcandidate therapeutic compositions, such as for example, 125P5C8intrabodies, 125P5C8 antisense molecules and ribozymes.

Example 16 125P5C8 Monoclonal Antibody-Mediated Inhibition of ProstateTumors In Vivo

The significant expression of 125P5C8, in cancer tissues, together withits restrictive expression in normal tissues along with its expectedcell surface expression makes 125P5C8 an excellent target for antibodytherapy. Similarly, 125P5C8 is a target for T cell-based immunotherapy.Thus, the therapeutic efficacy of anti-125P5C8 mAbs in human prostatecancer xenograft mouse models is evaluated by using androgen-independentLAPC-4 and LAPC-9 xenografts (Craft, N., et al., Cancer Res, 1999.59(19): p. 5030-6) and the androgen independent recombinant cell linePC3-125P5C8 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1):p. 16-23).

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in a mouse orthotopic prostate cancer xenograft model. Theantibodies can be unconjugated, as discussed in this Example, or can beconjugated to a therapeutic modality, as appreciated in the art. Wedemonstrate that anti-125P5C8 MAbs inhibit formation of both theandrogen-dependent LAPC-9 and androgen-independent PC3-125P5C8 tumorxenografts. Anti-125P5C8 mAbs also retard the growth of establishedorthotopic tumors and prolonged survival of tumor-bearing mice. Theseresults indicate the utility of anti-125P5C8 mAbs in the treatment oflocal and advanced stages of prostate cancer. (See, e.g., (Saffran, D.,et al., PNAS 10:1073-1078 orwww.pnas.org/cgi/doi/10.1073/pnas.051624698)

Administration of the anti-125P5C8 mAbs led to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 125P5C8 as anattractive target for immunotherapy and demonstrate the therapeuticpotential of anti-125P5C8 mAbs for the treatment of local and metastaticprostate cancer. This example demonstrates that unconjugated 125P5C8monoclonal antibodies are effective to inhibit the growth of humanprostate tumor xenografts grown in SCID mice; accordingly a combinationof such efficacious monoclonal antibodies is also effective.

Tumor Inhibition using Multiple Unconjugated 125P5C8 mAbs

Materials and Methods 125P5C8 Monoclonal Antibodies:

Monoclonal antibodies are raised against 125P5C8 as described in Example6. The antibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation for their capacity to bind 125P5C8. Epitope mappingdata for the anti-125P5C8 mAbs, as determined by ELISA and Westernanalysis, recognize epitopes on the 125P5C8 protein. Immunohistochemicalanalysis of prostate cancer tissues and cells with these antibodies isperformed.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofLAPC-9 prostate tumor xenografts.

Prostate Cancer Xenografts and Cell Lines.

The LAPC-9 xenograft, which expresses a wild-type androgen receptor andproduces prostate-specific antigen (PSA), is passaged in 6- to8-week-old male ICR-severe combined immunodeficient (SCID) mice (TaconicFarms) by s.c. trocar implant (Craft, N., et al., supra). Single-cellsuspensions of LAPC-9 tumor cells are prepared as described in Craft, etal. The prostate carcinoma cell line PC3 (American Type CultureCollection) is maintained in DMEM supplemented with L-glutamine and 10%(vol/vol) FBS.

A PC3-125P5C8 cell population is generated by retroviral gene transferas described in Hubert, R. S., et al., STEAP: a prostate-specificcell-surface antigen highly expressed in human prostate tumors. ProcNatl Acad Sci USA, 1999. 96(25): p. 14523-8. Anti-125P5C8 staining isdetected by using an FITC-conjugated goat anti-mouse antibody (SouthernBiotechnology Associates) followed by analysis on a Coulter Epics-XLflow cytometer.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ LAPC-9,PC3, or PC3-125P5C8 cells mixed at a 1:1 dilution with Matrigel(Collaborative Research) in the right flank of male SCID mice. To testantibody efficacy on tumor formation, i.p. antibody injections arestarted on the same day as tumor-cell injections. As a control, mice areinjected with either purified mouse IgG (ICN) or PBS; or a purifiedmonoclonal antibody that recognizes an irrelevant antigen not expressedin human cells. In preliminary studies, no difference is found betweenmouse IgG or PBS on tumor growth. Tumor sizes are determined by verniercaliper measurements, and the tumor volume is calculated aslength×width×height. Mice with s.c. tumors greater than 1.5 cm indiameter are sacrificed. PSA levels are determined by using a PSA ELISAkit (Anogen, Mississauga, Ontario). Circulating levels of anti-125P5C8mAbs are determined by a capture ELISA kit (Bethyl Laboratories,Montgomery, Tex.). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078or www.pnas.org/cgi/doi/10.1073/pnas.051624698)

Orthotopic injections are performed under anesthesia by usingketamine/xylazine. An incision is made through the abdominal muscles toexpose the bladder and seminal vesicles, which then are deliveredthrough the incision to expose the dorsal prostate. LAPC-9 cells (5×10⁵)mixed with Matrigel are injected into each dorsal lobe in a 10-μlvolume. To monitor tumor growth, mice are bled on a weekly basis fordetermination of PSA levels. Based on the PSA levels, the mice aresegregated into groups for the appropriate treatments. To test theeffect of anti-125P5C8 mAbs on established orthotopic tumors, i.p.antibody injections are started when PSA levels reach 2-80 ng/ml.

Anti-125P5C8 mAbs Inhibit Growth of 125P5C8-Expressing Prostate-CancerTumors

We next test the effect of anti-125P5C8 mAbs on tumor formation by usingthe LAPC-9 orthotopic model. As compared with the s.c. tumor model, theorthotopic model, which requires injection of tumor cells directly inthe mouse prostate, results in a local tumor growth, development ofmetastasis in distal sites, deterioration of mouse health, andsubsequent death (Saffran, D., et al., PNAS supra; Fu, X., et al., Int JCancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1):p. 4-8). The features make the orthotopic model more representative ofhuman disease progression and allowed us to follow the therapeuticeffect of mAbs on clinically relevant end points.

Accordingly, LAPC-9 tumor cells are injected into the mouse prostate,and 2 days later, the mice are segregated into two groups and treatedwith either up to 200 μg, usually 10-50 μg, of anti-125P5C8 Ab or PBSthree times per week for two to five weeks. Mice are monitored weeklyfor circulating PSA levels as an indicator of tumor growth.

A major advantage of the orthotopic prostate-cancer model is the abilityto study the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against a prostate-specific cell-surfaceprotein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R.S., et al., Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8).

Mice bearing established orthotopic LAPC-9 tumors are administered 11injections of either anti-125P5C8 mAb or PBS over a 4-week period. Micein both groups are allowed to establish a high tumor burden (PSA levelsgreater than 300 ng/ml), to ensure a high frequency of metastasisformation in mouse lungs. Mice then are killed and their prostate andlungs are analyzed for the presence of LAPC-9 cells by anti-STEAP IHCanalysis.

These studies demonstrate a broad anti-tumor efficacy of anti-125P5C8antibodies on initiation and progression of prostate cancer in xenograftmouse models. Anti-125P5C8 antibodies inhibit tumor formation of bothandrogen-dependent and androgen-independent tumors as well as retardingthe growth of already established tumors and prolong the survival oftreated mice. Moreover, anti-125P5C8 mAbs demonstrate a dramaticinhibitory effect on the spread of local prostate tumor to distal sites,even in the presence of a large tumor burden. Thus, anti-125P5C8 mAbsare efficacious on major clinically relevant end points/PSA levels(tumor growth), prolongation of survival, and health.

Example 17 Androgen Regulation of 125P5C8

Since 125P5C8 was derived from a LAPC-9 AD (14 days post-castration)minus LAPC-9 AD (no castration) subtraction, androgen regulation of125P5C8 expression is studied (FIG. 11). LAPC-4 AD and LAPC-9 AD cellsare grown in charcoal-stripped medium and stimulated with the syntheticandrogen mibolerone, for either 14 or 24 hours. Expression of 125P5C8 isstudied before and after stimulation with mibolerone. The experimentalsamples are confirmed by testing for the expression of theandrogen-regulated prostate cancer gene PSA. In another experiment,125P5C8 expression is analyzed in LAPC-9 AD and LAPC-9 AI tumors grownin castrated mice. Only, androgen independent tumors will grow incastrated mice.

When 125P5C8 expression is regulated by androgen, 125P5C8 is a targetfor diagnostic, preventative and therapeutic purposes.

Throughout this application, various publications and applications arereferenced (within parentheses for example). The disclosures of thesepublications and applications are hereby incorporated by referenceherein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Tables

TABLE I Tissues that Express 125P5C8 When Malignant Prostate BladderKidney Colon

TABLE II AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III AMINO ACID SUBSTITUTION MATRIX Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins. $\begin{matrix}A & C & D & E & F & G & H & I & K & L & M & N & P & Q & R & S & T & V & W & Y & \; \\4 & 0 & {- 2} & {- 1} & {- 2} & 0 & {- 2} & {- 1} & {- 1} & {- 1} & {- 1} & {- 2} & {- 1} & {- 1} & {- 1} & 1 & 0 & 0 & {- 3} & {- 2} & A \\\; & 9 & {- 3} & {- 4} & {- 2} & {- 3} & {- 3} & {- 1} & {- 3} & {- 1} & {- 1} & {- 3} & {- 3} & {- 3} & {- 3} & {- 1} & {- 1} & {- 1} & {- 2} & {- 2} & C \\\; & \; & 6 & 2 & {- 3} & {- 1} & {- 1} & {- 3} & {- 1} & {- 4} & {- 3} & 1 & {- 1} & 0 & {- 2} & 0 & {- 1} & {- 3} & {- 4} & {- 3} & D \\\; & \; & \; & 5 & {- 3} & {- 2} & 0 & {- 3} & 1 & {- 3} & {- 2} & 0 & {- 1} & 2 & 0 & 0 & {- 1} & {- 2} & {- 3} & {- 2} & E \\\; & \; & \; & \; & 6 & {- 3} & {- 1} & 0 & {- 3} & 0 & 0 & {- 3} & {- 4} & {- 3} & {- 3} & {- 2} & {- 2} & {- 1} & 1 & 3 & F \\\; & \; & \; & \; & \; & 6 & {- 2} & {- 4} & {- 2} & {- 4} & {- 3} & 0 & {- 2} & {- 2} & {- 2} & 0 & {- 2} & {- 3} & {- 2} & {- 3} & G \\\; & \; & \; & \; & \; & \; & 8 & {- 3} & {- 1} & {- 3} & {- 2} & 1 & {- 2} & 0 & 0 & {- 1} & {- 2} & {- 3} & {- 2} & 2 & H \\\; & \; & \; & \; & \; & \; & \; & 4 & {- 3} & 2 & 1 & {- 3} & {- 3} & {- 3} & {- 3} & {- 2} & {- 1} & 3 & {- 3} & {- 1} & I \\\; & \; & \; & \; & \; & \; & \; & \; & 5 & {- 2} & {- 1} & 0 & {- 1} & 1 & 2 & 0 & {- 1} & {- 2} & {- 3} & {- 2} & K \\\; & \; & \; & \; & \; & \; & \; & \; & \; & 4 & 2 & {- 3} & {- 3} & {- 2} & {- 2} & {- 2} & {- 1} & 1 & {- 2} & {- 1} & L \\\; & \; & \; & \; & \; & \; & \; & \; & \; & \; & 5 & {- 2} & {- 2} & 0 & {- 1} & {- 1} & {- 1} & 1 & {- 1} & {- 1} & M \\\; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \mspace{11mu} & 6 & {- 2} & 0 & 0 & 1 & 0 & {- 3} & {- 4} & {- 2} & N \\\; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & 7 & {- 1} & {- 2} & {- 1} & {- 1} & {- 2} & {- 4} & {- 3} & P \\\; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & 5 & 1 & 0 & {- 1} & {- 2} & {- 2} & {- 1} & Q \\\; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & 5 & {- 1} & {- 1} & {- 3} & {- 3} & {- 2} & R \\\; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & 4 & 1 & {- 2} & {- 3} & {- 2} & S \\\; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & 5 & 0 & {- 2} & {- 2} & T \\\mspace{14mu} & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & 4 & {- 3} & {- 1} & V \\\; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & 11 & 2 & W \\\; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & \; & 7 & {Y\quad}\end{matrix}\quad$

TABLE IV (A) HLA CLASS I SUPERMOTIFS SUPERMOTIF POSITION 2 C-TERMINUS A2L, I, V, M, A, T, Q L,. I, V, M, A, T A3 A, V, I, L, M, S, T R, K B7 PA, L, I, M, V, F, W, Y B44 D, E F, W, Y, L, I, M, V, A A1 T, S, L, I, V,M F, W, Y A24 F, W, Y, L, V, I, M, T F, I, Y, W, L, M B27 R, H, K A, L,I, V, M, Y, F, W B58 A, S, T F, W, Y, L, I, V B62 L, V, M, P, I, Q F, W,Y, M, I, V

TABLE IV (B) HLA CLASS II SUPERMOTIF 1 6 9 W, F, Y, V, .I, L A, V, I, L,P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE V Peptide Scoring Results - 125P5C8 A1 9-mers Score (Estimate ofHalf Subsequence Time of Disassociation of Start Residue A MoleculeContaining Rank Position Listing This Subsequence)  1 41 GLEGFSIAF45.000  2 635 DSEIQMAKF 27.000  3 490 YTDFGPSTR 12.500  4 371 NLDLLLQTK10.000  5 583 TSAPGSRDY 7.500  6 514 KSEHHLLPS 6.750  7 231 GPDPNPFGG6.250  8 22 YHDLGPMIY 6.250  9 602 DIDSTDHDR 5.000 10 541 LVDFVVTHF5.000 11 213 FGEVSLVSR 4.500 12 36 TLELTGLEG 4.500 13 249 LMLPSCLWF2.500 14 269 TASAAGLLY 2.500 15 132 VLVVLRIWY 2.500 16 431 AIWPFRFGY2.500 17 24 DLGPMIYYF 2.000 18 611 WCEYIMYRG 1.800 19 466 ESDASKPYM1.500 20 388 KSEKYMKLF 1.350 21 315 TMTIAMIFY 1.250 22 314 KTMTIAMIF1.250 23 645 IPDDPTNYR 1.250 24 562 AIAVSKLLK 1.000 25 413 KAYERKLGK1.000 26 54 FLTITPFWK 1.000 27 9 LLESLLGCV 0.900 28 324 LLEIFFCAW 0.90029 551 NHEDDLDRK 0.900 30 630 HAELSDSEI 0.900 31 159 LSAIATLDR 0.750 32141 TSLNPIWSY 0.750 33 348 RSDVLLGTM 0.750 34 112 WSGSHLQRY 0.750 35 633LSDSEIQMA 0.750 36 573 SNQVIFLGY 0.625 37 358 LIIGLNMLF 0.500 38 49FLSPIFLTI 0.500 39 429 SAAIWPFRF 0.500 40 644 RIPDDPTNY 0.500 41 407GLGLRHKAY 0.500 42 482 WLGEKLGFY 0.500 43 614 YIMYRGLIR 0.500 44 76ITIGSIASF 0.500 45 199 GAAFGSLVF 0.500 46 594 LTEHGNVKD 0.450 47 524EGEIAPAIT 0.450 48 522 SPEGEIAPA 0.450 49 559 KLQAIAVSK 0.400 50 463TILESDASK 0.400

TABLE VI HLA Peptide Scoring Results-125P5C8-A1 10-mers Score (Estimateof Half Subsequence Time of Disassociation of Start Residue A MoleculeContaining Rank Position Listing This Subsequence)  1 633 LSDSEIQMAK75.000  2 605 STDHDRWCEY 62.500  3 490 YTDFGPSTRY 62.500  4 464ILESDASKPY 45.000  5 635 DSEIQMAKFR 13.500  6 440 DNEGWSSLER 11.250  7659 VIDHREVSEK 10.000  8 36 TLELTGLEGF 9.000  9 22 YHDLGPMIYY 6.250 10268 GTASAAGLLY 6.250 11 314 KTMTIAMIFY 6.250 12 572 SSNQVIFLGY 3.750 13171 DGDCSKPEEK 2.500 14 430 AAIWPFRFGY 2.500 15 131 IVLVVLRIWY 2.500 16458 GADFITILES 2.500 17 662 HREVSEKIHF 2.250 18 594 LTEHGNVKDI 2.250 1941 GLEGFSIAFL 1.800 20 324 LLEIFFCAWC 1.800 21 466 ESDASKPYMG 1.500 22665 VSEKIHFNPR 1.350 23 140 YTSLNPIWSY 1.250 24 309 GTNPGKTMTI 1.250 25582 ITSAPGSRDY 1.250 26 231 GPDPNPFGGA 1.250 27 524 EGEIAPAITL 1.125 28182 TGEVATGMAS 1.125 29 454 LNETGADFIT 1.125 30 57 ITPFWKLVNK 1.000 31505 MALSRYPIVK 1.000 32 561 QAIAVSKLLK 1.000 33 462 ITILESDASK 1.000 349 LLESLLGCVS 0.900 35 630 HAELSDSEIQ 0.900 36 611 WCEYIMYRGL 0.900 37428 VSAAIWPFRF 0.750 38 348 RSDVLLGTMM 0.750 39 388 KSEKYMKLFL 0.675 40329 FCAWCTAFKF 0.500 41 541 LVDFVVTHFG 0.500 42 158 TLSAIATLDR 0.500 43531 ITLTVNISGK 0.500 44 320 MIFYLLEIFF 0.500 45 13 LLGCVSWSLY 0.500 46371 NLDLLLQTKN 0.500 47 56 TITPFWKLVN 0.500 48 526 EIAPAITLTV 0.500 49383 KVLFRKSEKY 0.500 50 357 MLIIGLNMLF 0.500

TABLE VII HLA Peptide Scoring Results - 125P5C8- A2 9-mers Score(Estimate of Half Subsequence Time of Disassociation of Start Residue AMolecule Containing Rank Position Listing This Subsequence)  1 323YLLEIFFCA 3820.380  2 62 KLVNKKWML 560.763  3 204 SLVFLTHWV 382.536  4126 FILGQIVLV 374.369  5 277 YLHTWAAAV 319.939  6 8 ILLESLLGC 294.675  713 LLGCVSWSL 272.371  8 92 RLMVLALGV 257.342  9 211 WVFGEVSLV 238.235 10275 LLYLHTWAA 202.694 11 615 IMYRGLIRL 193.040 12 254 CLWFRGTGL 177.30813 392 YMKLFLWLL 162.824 14 351 VLLGTMMLI 150.931 15 364 MLFGPKKNL134.369 16 241 VLLCLASGL 134.369 17 127 ILGQIVLVV 111.499 18 398WLLVGVGLL 108.713 19 133 LVVLRIWYT 105.168 20 274 GLLYLHTWA 101.099 2149 FLSPIFLTI 91.183 22 188 GMASRPNWL 84.856 23 357 MLIIGLNML 83.527 2456 TITPFWKLV 61.780 25 258 RGTGLIWWV 43.075 26 316 MTIAMIFYL 37.007 2768 WMLTLLRII 24.186 28 356 MMLIIGLNM 22.569 29 216 VSLVSRWAV 21.418 3028 MIYYFPLQT 21.182 31 120 YLRIWGFIL 17.760 32 319 AMIFYLLEI 17.330 33261 GLIWWVTGT 17.140 34 352 LLGTMMLII 16.725 35 473 YMGNNDLTM 16.505 36149 YQMSNKVIL 15.114 37 200 AAFGSLVFL 13.887 38 90 KLRLMVLAL 13.070 39504 IMALSRYPI 12.809 40 156 ILTLSAIAT 12.668 41 150 QMSNKVILT 12.379 42284 AVSGCVFAI 12.178 43 376 LQTKNSSKV 11.988 44 97 ALGVSSSLI 10.433 4547 IAFLSPIFL 10.264 46 540 KLVDFVVTH 9.346 47 42 LEGFSIAFL 8.933 48 560LQAIAVSKL 8.469 49 34 LQTLELTGL 8.469 50 154 KVILTLSAI 7.349

TABLE VIII HLA Peptide Scoring Results - 125P5C8 A2 10-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence)  1 482WLGEKLGFYT 4483.377  2 394 KLFLWLLVGV 2071.606  3 54 FLTITPFWKL 1400.305 4 132 VLVVLRIWYT 1201.914  5 315 TMTIAMIFYL 1131.982  6 567 KLLKSSSNQV900.698  7 396 FLWLLVGVGL 815.616  8 12 SLLGCVSWSL 592.807  9 8ILLESLLGCV 536.309 10 356 MMLIIGLNML 223.203 11 453 LLNETGADFI 195.97112 559 KLQAIAVSKL 171.967 13 384 VLFRKSEKYM 171.868 14 126 FILGQIVLVV153.491 15 274 GLLYLHTWAA 137.862 16 49 FLSPIFLTIT 122.836 17 375LLQTKNSSKV 118.238 18 188 GMASRPNWLL 115.713 19 614 YIMYRGLIRL 114.98520 330 CAWCTAFKFV 83.786 21 399 LLVGVGLLGL 83.527 22 156 ILTLSAIATL83.527 23 207 FLTHWVFGEV 79.025 24 351 VLLGTMMLII 61.882 25 536NISGKLVDFV 59.279 26 363 NMLFGPKKNL 57.085 27 504 IMALSRYPIV 52.518 28275 LLYLHTWAAA 45.944 29 62 KLVNKKWMLT 44.339 30 591 YLQLTEHGNV 41.59231 69 MLTLLRIITI 40.792 32 296 SMWPQTLGHL 38.289 33 68 WMLTLLRIIT 37.55734 323 YLLEIFFCAW 37.545 35 28 MIYYFPLQTL 36.752 36 242 LLCLASGLML36.316 37 95 VLALGVSSSL 36.316 38 150 QMSNKVILTL 35.485 39 127ILGQIVLVVL 34.246 40 20 SLYHDLGPMI 33.385 41 149 YQMSNKVILT 29.577 42 97ALGVSSSLIV 28.516 43 137 RIWYTSLNPI 27.385 44 342 GVYARERSDV 19.475 45134 VVLRIWYTSL 17.636 46 41 GLEGFSIAFL 17.295 47 46 SIAFLSPIFL 16.155 48619 GLIRLGYARI 15.649 49 392 YMKLFLWLLV 13.748 50 355 TMMLIIGLNM 13.276

TABLE IX HLA Peptide Scoring Results - 125P5C8 A3 9-mers Score (Estimateof Half Time of Subsequence Disassociation of a Start Residue MoleculeContaining Rank Position Listing This Subsequence)  1 506 ALSRYPIVK120.000  2 418 KLGKVAPTK 90.000  3 559 KLQAIAVSK 90.000  4 54 FLTITPFWK60.000  5 619 GLIRLGYAR 54.000  6 361 GLNMLFGPK 54.000  7 41 GLEGFSIAF54.000  8 409 GLRHKAYER 36.000  9 371 NLDLLLQTK 30.000 10 532 TLTVNISGK30.000 11 593 QLTEHGNVK 30.000 12 431 AIWPFRFGY 27.000 13 384 VLFRKSEKY20.000 14 375 LLQTKNSSK 20.000 15 250 MLPSCLWFR 18.000 16 315 TMTIAMIFY12.000 17 132 VLVVLRIWY 12.000 18 90 KLRLMVLAL 10.800 19 413 KAYERKLGK9.000 20 615 IMYRGLIRL 9.000 21 249 LMLPSCLWF 9.000 22 383 KVLFRKSEK9.000 23 392 YMKLFLWLL 8.100 24 319 AMIFYLLEI 8.100 25 540 KLVDFVVTH8.100 26 478 DLTMWLGEK 8.100 27 62 KLVNKKWML 8.100 28 49 FLSPIFLTI 8.10029 323 YLLEIFFCA 6.075 30 407 GLGLRHKAY 6.000 31 338 FVPGGVYAR 5.400 32120 YLRIWGFIL 5.400 33 463 TILESDASK 4.500 34 24 DLGPMIYYF 4.050 35 351VLLGTMMLI 4.050 36 261 GLIWWVTGT 4.050 37 562 AIAVSKLLK 4.000 38 364MLFGPKKNL 3.375 39 275 LLYLHTWAA 3.000 40 453 LLNETGADF 3.000 41 205LVFLTHWVF 3.000 42 405 LLGLGLRHK 3.000 43 296 SMWPQTLGH 3.000 44 254CLWFRGTGL 3.000 45 691 HMNTPKYFL 2.700 46 404 GLLGLGLRH 2.700 47 482WLGEKLGFY 2.700 48 13 LLGCVSWSL 2.700 49 394 KLFLWLLVG 2.700 50 188GMASRPNWL 1.800

TABLE X HLA Peptide Scoring Results - 125P5C8 A3 10-mers Score (Estimateof Half Time of Subsequence Disassociation of a Start Residue MoleculeContaining Rank Position Listing This Subsequence)  1 361 GLNMLFGPKK180.000  2 409 GLRHKAYERK 60.000  3 540 KLVDFVVTHF 40.500  4 249LMLPSCLWFR 40.500  5 374 LLLQTKNSSK 30.000  6 404 GLLGLGLRHK 20.250  7480 TMWLGEKLGF 20.000  8 248 GLMLPSCLWF 18.000  9 204 SLVFLTHWVF 9.00010 188 GMASRPNWLL 8.100 11 54 FLTITPFWKL 8.100 12 158 TLSAIATLDR 8.00013 12 SLLGCVSWSL 6.075 14 659 VIDHREVSEK 6.000 15 357 MLIIGLNMLF 6.00016 559 KLQAIAVSKL 5.400 17 394 KLFLWLLVGV 4.500 18 396 FLWLLVGVGL 4.50019 319 AMIFYLLEIF 4.500 20 351 VLLGTMMLII 4.050 21 323 YLLEIFFCAW 4.05022 399 LLVGVGLLGL 4.050 23 41 GLEGFSIAFL 4.050 24 13 LLGCVSWSLY 4.000 2520 SLYHDLGPMI 3.000 26 452 HLLNETGADF 3.000 27 500 HTWGIMALSR 3.000 2836 TLELTGLEGF 3.000 29 58 TPFWKLVNKK 3.000 30 150 QMSNKVILTL 2.700 31619 GLIRLGYARI 2.700 32 315 TMTIAMIFYL 2.700 33 142 SLNPIWSYQM 2.700 34274 GLLYLHTWAA 2.700 35 314 KTMTIAMIFY 2.700 36 531 ITLTVNISGK 2.250 37296 SMWPQTLGHL 2.025 38 167 RIGTDGDCSK 2.000 39 464 ILESDASKPY 2.000 40320 MIFYLLEIFF 2.000 41 305 LINSGTNPGK 2.000 42 383 KVLFRKSEKY 1.800 43505 MALSRYPIVK 1.800 44 69 MLTLLRIITI 1.800 45 145 PIWSYQMSNK 1.500 4657 ITPFWKLVNK 1.500 47 462 ITILESDASK 1.500 48 127 ILGQIVLVVL 1.350 49284 AVSGCVFAIF 1.350 50 140 YTSLNPIWSY 1.350

TABLE XI HLA Peptide Scoring Results - 125P5C8 A11 9-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence)  1 383KYLFRKSEK 9.000  2 413 KAYERKLGK 2.400  3 54 FLTITPFWK 1.200  4 418KLGKVAPTK 1.200  5 361 GLNMLFGPK 1.200  6 559 KLQAIAVSK 1.200  7 506ALSRYPIVK 0.800  8 562 AIAVSKLLK 0.800  9 338 FVPGGVYAR 0.800 10 619GLIRLGYAR 0.720 11 463 TILESDASK 0.600 12 129 GQIVLVVLR 0.540 13 409GLRHKAYER 0.480 14 371 NLDLLLQTK 0.400 15 532 TLTVNISGK 0.400 16 375LLQTKNSSK 0.400 17 58 TPFWKLVNK 0.400 18 593 QLTEHGNVK 0.400 19 614YIMYRGLIR 0.320 20 329 FCAWCTAFK 0.200 21 490 YTDFGPSTR 0.200 22 250MLPSCLWFR 0.160 23 314 KTMTIAMIF 0.120 24 402 GVGLLGLGL 0.120 25 655NQKVVIDHR 0.120 26 478 DLTMWLGEK 0.120 27 354 GTMMLIIGL 0.120 28 184EVATGMASR 0.120 29 649 PTNYRDNQK 0.100 30 154 KVILTLSAI 0.090 31 581YITSAPGSR 0.080 32 362 LNMLFGPKK 0.080 33 205 LVFLTHWVF 0.080 34 380NSSKVLFRK 0.060 35 172 GDCSKPEEK 0.060 36 284 AVSGCVFAI 0.060 37 84FQAPNAKLR 0.060 38 173 DCSKPEEKK 0.060 39 550 GNHEDDLDR 0.048 40 66KKWMLTLLR 0.048 41 379 KNSSKVLFR 0.048 42 92 RLMVLALGV 0.048 43 391KYMKLFLWL 0.048 44 316 MTIAMIFYL 0.045 45 688 HHFHMNTPK 0.040 46 386FRKSEKYMK 0.040 47 671 FNPRFGSYK 0.040 48 405 LLGLGLRHK 0.040 49 400LVGVGLLGL 0.040 50 306 INSGTNPGK 0.040

TABLE XII HLA Peptide Scoring Results - 125P5C8 A11 10-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence)  1 462ITILESDASK 1.500  2 531 ITLTVNISGK 1.500  3 409 GLRHKAYERK 1.200  4 361GLNMLFGPKK 1.200  5 402 GVGLLGLGLR 1.200  6 167 RIGTDGDCSK 1.200  7 57ITPFWKLVNK 1.000  8 53 IFLTITPFWK 0.900  9 592 LQLTEHGNVK 0.900 10 500HTWGIMALSR 0.800 11 374 LLLQTKNSSK 0.600 12 561 QAIAVSKLLK 0.600 13 505MALSRYPIVK 0.600 14 305 LINSGTNPGK 0.400 15 58 TPFWKLVNKK 0.400 16 659VIDHREVSEK 0.400 17 385 LFRKSEKYMK 0.400 18 379 KNSSKVLFRK 0.360 19 337KFVPGGVYAR 0.360 20 580 GYITSAPGSR 0.360 21 249 LMLPSCLWFR 0.240 22 644RIPDDPTNYR 0.240 23 670 HFNPRFGSYK 0.200 24 328 FFCAWCTAFK 0.200 25 81IASFQAPNAK 0.200 26 370 KNLDLLLQTK 0.180 27 404 GLLGLGLRHK 0.180 28 158TLSAIATLDR 0.160 29 550 GNHEDDLDRK 0.120 30 342 GVYARERSDV 0.120 31 427EVSAAIWPFR 0.120 32 314 KTMTIAMIFY 0.120 33 558 RKLQAIAVSK 0.090 34 417RKLGKVAPTK 0.090 35 383 KVLFRKSEKY 0.090 36 154 KVILTLSAIA 0.090 37 145PIWSYQMSNK 0.080 38 489 FYTDFGPSTR 0.080 39 613 EYIMYRGLIR 0.072 40 172GDCSKPEEKK 0.060 41 268 GTASAAGLLY 0.060 42 421 KVAPTKEVSA 0.060 43 131IVLVVLRIWY 0.060 44 648 DPTNYRDNQK 0.060 45 99 GVSSSLIVQA 0.060 46 309GTNPGKTMTI 0.060 47 129 GQIVLVVLRI 0.054 48 119 RYLRIWGFIL 0.054 49 183GEVATGMASR 0.054 50 248 GLMLPSCLWF 0.048

TABLE XIII HLA Peptide Scoring Results - 125P5C8 A24 9-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence)  1 391KYMKLFLWL 864.000  2 29 IYYFPLQTL 240.000  3 119 RYLRIWGFI 210.000  4148 SYQMSNKVI 75.000  5 613 EYIMYRGLI 75.000  6 21 LYHDLGPMI 72.000  731 YFPLQTLEL 33.000  8 83 SFQAPNAKL 33.000  9 125 GFILGQIVL 30.000 10548 HFGNHEDDL 20.000 11 62 KLVNKKWML 12.000 12 321 IFYLLEIFF 12.000 13328 FFCAWCTAF 10.000 14 498 RYHTWGIMA 10.000 15 471 KPYMGNNDL 9.600 16533 LTVNISGKL 9.240 17 475 GNNDLTMWL 8.640 18 314 KTMTIAMIF 8.400 19 96LALGVSSSL 8.400 20 151 MSNKVILTL 8.400 21 397 LWLLVGVGL 8.400 22 561QAIAVSKLL 8.400 23 128 LGQIVLVVL 8.400 24 414 AYERKLGKV 8.250 25 90KLRLMVLAL 8.000 26 55 LTITPFWKL 7.920 27 479 LTMWLGEKL 7.920 28 276LYLHTWAAA 7.500 29 580 GYITSAPGS 7.500 30 322 FYLLEIFFC 7.500 31 247SGLMLPSCL 7.200 32 445 SSLERSAHL 7.200 33 357 MLIIGLNML 7.200 34 241VLLCLASGL 7.200 35 354 GTMMLIIGL 7.200 36 317 TIAMIFYLL 6.720 37 85QAPNAKLRL 6.000 38 388 KSEKYMKLF 6.000 39 584 SAPGSRDYL 6.000 40 243LCLASGLML 6.000 41 149 YQMSNKVIL 6.000 42 26 GPMIYYFPL 6.000 43 297MWPQTLGHL 6.000 44 366 FGPKKNLDL 6.000 45 157 LTLSAIATL 6.000 46 350DVLLGTMML 6.000 47 489 FYTDFGPST 6.000 48 691 HMNTPKYFL 6.000 49 210HWVFGEVSL 6.000 50 139 WYTSLNPIW 6.000

TABLE XIV HLA Peptide Scoring Results - 125P5C8 A24 10-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence)  1 391KYMKLFLWLL 600.000  2 119 RYLRIWGFIL 600.000  3 498 RYHTWGIMAL 400.000 4 148 SYQMSNKVIL 300.000  5 30 YYFPLQTLEL 264.000  6 624 GYARISHAEL220.000  7 343 VYARERSDVL 200.000  8 438 GYDNEGWSSL 200.000  9 651NYRDNQKVVI 60.000 10 683 NYENNHHFHM 37.500 11 365 LFGPKKNLDL 24.000 12559 KLQAIAVSKL 13.200 13 322 FYLLEIFFCA 12.600 14 48 AFLSPIFLTI 12.60015 388 KSEKYMKLFL 12.000 16 316 MTIAMIFYLL 10.080 17 540 KLVDFVVTHF10.080 18 689 HFHMNTPKYF 10.000 19 327 IFFCAWCTAF 10.000 20 414AYERKLGKVA 9.000 21 12 SLLGCVSWSL 8.400 22 570 KSSSNQVIFL 8.000 23 590DYLQLTEHGN 7.500 24 276 LYLHTWAAAV 7.500 25 401 VGVGLLGLGL 7.200 26 445SSLERSAHLL 7.200 27 474 MGNNDLTMWL 7.200 28 187 TGMASRPNWL 7.200 29 233DPNPFGGAVL 7.200 30 240 AVLLCLASGL 7.200 31 356 MMLIIGLNML 7.200 32 616MYRGLIRLGY 7.000 33 677 SYKEGHNYEN 6.600 34 532 TLTVNISGKL 6.160 35 614YIMYRGLIRL 6.000 36 611 WCEYIMYRGL 6.000 37 363 NMLFGPKKNL 6.000 38 510YPIVKSEHHL 6.000 39 63 LVNKKWMLTL 6.000 40 21 LYHDLGPMIY 6.000 41 399LLVGVGLLGL 6.000 42 397 LWLLVGVGLL 6.000 43 134 VVLRIWYTSL 6.000 44 366FGPKKNLDLL 6.000 45 524 EGEIAPAITL 6.000 46 25 LGPMIYYFPL 6.000 47 253SCLWFRGTGL 6.000 48 41 GLEGFSIAFL 6.000 49 4 LWREILLESL 5.760 50 127ILGQIVLVVL 5.600

TABLE XV HLA Peptide Scoring Results - 125P5C8 B7 9-mers Score (Estimateof Half Time of Subsequence Disassociation of a Start Residue MoleculeContaining Rank Position Listing This Subsequence)  1 26 GPMIYYFPL240.000  2 344 YARERSDVL 120.000  3 625 YARISHAEL 120.000  4 367GPKKNLDLL 80.000  5 235 NPFGGAVLL 80.000  6 471 KPYMGNNDL 80.000  7 86APNAKLRLM 60.000  8 90 KLRLMVLAL 40.000  9 120 YLRIWGFIL 40.000 10 135VLRIWYTSL 40.000 11 200 AAFGSLVFL 36.000 12 402 GVGLLGLGL 20.000 13 350DVLLGTMML 20.000 14 400 LVGVGLLGL 20.000 15 512 IVKSEHHLL 20.000 16 189MASRPNWLL 18.000 17 584 SAPGSRDYL 18.000 18 270 ASAAGLLYL 12.000 19 85QAPNAKLRL 12.000 20 197 LAGAAFGSL 12.000 21 47 IAFLSPIFL 12.000 22 561QAIAVSKLL 12.000 23 354 GTMMLIIGL 12.000 24 294 TASMWPQTL 12.000 25 149YQMSNKVIL 12.000 26 479 LTMWLGEKL 12.000 27 96 LALGVSSSL 12.000 28 88NAKLRLMVL 12.000 29 298 WPQTLGHLI 8.000 30 55 LTITPFWKL 6.000 31 691HMNTPKYFL 6.000 32 1 MTSLWREIL 6.000 33 364 MLFGPKKNL 6.000 34 284AVSGCVFAI 6.000 35 423 APTKEVSAA 6.000 36 64 VNKKWMLTL 4.000 37 392YMKLFLWLL 4.000 38 254 CLWFRGTGL 4.000 39 2 TSLWREILL 4.000 40 366FGPKKNLDL 4.000 41 571 SSSNQVIFL 4.000 42 151 MSNKVILTL 4.000 43 109VTWWSGSHL 4.000 44 357 MLIIGLNML 4.000 45 620 LIRLGYARI 4.000 46 237FGGAVLLCL 4.000 47 34 LQTLELTGL 4.000 48 128 LGQIVLVVL 4.000 49 268GTASAAGLL 4.000 50 316 MTIAMIFYL 4.000

TABLE XVI HLA Peptide Scoring Results 125P5C8 B7 10-mers Score (Estimateof Half Time of Subsequence Disassociation of a Start Residue MoleculeContaining Rank Position Listing This Subsequence)  1 585 APGSRDYLQL240.000  2 344 YARERSDVLL 120.000  3 510 YPIVKSEHHL 80.000  4 367GPKKNLDLLL 80.000  5 233 DPNPFGGAVL 80.000  6 108 AVTWWSGSHL 60.000  7240 AVLLCLASGL 60.000  8 528 APAITLTVNI 24.000  9 423 APTKEVSAAI 24.00010 134 VVLRIWYTSL 20.000 11 311 NPGKTMTIAM 20.000 12 16 CVSWSLYHDL20.000 13 63 LVNKKWMLTL 20.000 14 86 APNAKLRLMV 18.000 15 82 ASFQAPNAKL18.000 16 187 TGMASRPNWL 12.000 17 246 ASGLMLPSCL 12.000 18 269TASAAGLLYL 12.000 19 199 GAAFGSLVFL 12.000 20 614 YIMYRGLIRL 12.000 21496 STRYHTWGIM 10.000 22 188 GMASRPNWLL 6.000 23 363 NMLFGPKKNL 6.000 2428 MIYYFPLQTL 6.000 25 583 TSAPGSRDYL 6.000 26 54 FLTITPFWKL 6.000 27288 CVFAIFTASM 5.000 28 560 LQAIAVSKLL 4.000 29 353 LGTMMLIIGL 4.000 304 LWREILLESL 4.000 31 1 MTSLWREILL 4.000 32 84 FQAPNAKLRL 4.000 33 253SCLWFRGTGL 4.000 34 242 LLCLASGLML 4.000 35 570 KSSSNQVIFL 4.000 36 399LLVGVGLLGL 4.000 37 293 FTASMWPQTL 4.000 38 396 FLWLLVGVGL 4.000 39 127ILGQIVLVVL 4.000 40 266 VTGTASAAGL 4.000 41 12 SLLGCVSWSL 4.000 42 532TLTVNISGKL 4.000 43 376 LQTKNSSKVL 4.000 44 150 QMSNKVILTL 4.000 45 124WGFILGQIVL 4.000 46 46 SIAFLSPIFL 4.000 47 366 FGPKKNLDLL 4.000 48 474MGNNDLTMWL 4.000 49 112 WSGSHLQRYL 4.000 50 315 TMTIAMIFYL 4.000

TABLE XVII HLA Peptide Scoring Results - 125P5C8 B35 9-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence)  1 367GPKKNLDLL 60.000  2 86 APNAKLRLM 40.000  3 471 KPYMGNNDL 40.000  4 229HPGPDPNPF 30.000  5 26 GPIYYFPL 20.000  6 235 NPFGGAVLL 20.000  7 344YARERSDVL 18.000  8 676 GSYKEGHNY 15.000  9 644 RIPDDPTNY 12.000 10 112WSGSHLQRY 10.000 11 445 SSLERSAHL 10.000 12 494 GPSTRYHTW 10.000 13 583TSAPGSRDY 10.000 14 141 TSLNPIWSY 10.000 15 570 KSSSNQVIF 10.000 16 88NAKLRLMVL 9.000 17 625 YARISHAEL 9.000 18 298 WPQTLGHLI 8.000 19 181KTGEVATGM 8.000 20 90 KLRLMVLAL 6.000 21 348 RSDVLLGTM 6.000 22 269TASAAGLLY 6.000 23 17 VSWSLYHDL 5.000 24 2 TSLWREILL 5.000 25 270ASAAGLLYL 5.000 26 571 SSSNQVIFL 5.000 27 285 VSGCVFAIF 5.000 28 151MSNKVILTL 5.000 29 512 IVKSEHHLL 4.500 30 192 RPNWLLAGA 4.000 31 632ELSDSEIQM 4.000 32 233 DPNPFGGAV 4.000 33 482 WLGEKLGFY 4.000 34 197LAGAAFGSL 3.000 35 96 LALGVSSSL 3.000 36 330 CAWCTAFKF 3.000 37 520LPSPEGEIA 3.000 38 423 APTKEVSAA 3.000 39 120 YLRIWGFIL 3.000 40 466ESDASKPYM 3.000 41 64 VNKKWMLTL 3.000 42 85 QAPNAKLRL 3.000 43 392YMKLFLWLL 3.000 44 561 QAIAVSKLL 3.000 45 587 GSRDYLQLT 3.000 46 377QTKNSSKVL 3.000 47 388 KSEKYMKLF 3.000 48 294 TASMWPQTL 3.000 49 135VLRIWYTSL 3.000 50 199 GAAFGSLVF 3.000

TABLE XVIII HLA Peptide Scoring Results - 125P5C8 B35 10-mers Score(Estimate of Half Time of Subsequence Disassociation of a Start ResidueMolecule Containing Rank Position Listing This Subsequence)  1 367GPKKNLDLLL 60.000  2 311 NPGKTMTIAM 40.000  3 585 APGSRDYLQL 30.000  4233 DPNPFGGAVL 20.000  5 510 YPIVKSEHHL 20.000  6 51 SPIFLTITPF 20.000 7 344 YARERSDVLL 18.000  8 19 WSLYHDLGPM 15.000  9 307 NSGTNPGKTM10.000 10 445 SSLERSAHLL 10.000 11 572 SSNQVIFLGY 10.000 12 570KSSSNQVIFL 10.000 13 528 APAITLTVNI 8.000 14 423 APTKEVSAAI 8.000 15 430AAIWPFRFGY 6.000 16 496 STRYHTWGIM 6.000 17 348 RSDVLLGTMM 6.000 18 85QAPNAKLRLM 6.000 19 428 VSAAIWPFRF 5.000 20 45 FSIAFLSPIF 5.000 21 246ASGLMLPSCL 5.000 22 583 TSAPGSRDYL 5.000 23 444 WSSLERSAHL 5.000 24 112WSGSHLQRYL 5.000 25 829 ASFQAPNAKL 5.000 26 176 KPEEKKTGEV 4.800 27 383KVLFRKSEKY 4.000 28 471 KPYMGNNDLT 4.000 29 314 KTMTIAMIFY 4.000 30 540KLVDFVVTHF 4.000 31 86 APNAKLRLMV 4.000 32 192 RPNWLLAGAA 4.000 33 281WAAAVSGCVF 3.000 34 117 LQRYLRIWGF 3.000 35 377 QTKNSSKVLF 3.000 36 199GAAFGSLVFL 3.000 37 388 KSEKYMKLFL 3.000 38 64 VNKKWMLTLL 3.000 39 269TASAAGLLYL 3.000 40 675 FGSYKEGHNY 3.000 41 102 SSLIVQAVTW 2.500 42 522SPEGEIAPAI 2.400 43 413 KAYERKLGKV 2.400 44 131 IVLVVLRIWY 2.000 45 235NPFGGAVLLC 2.000 46 355 TMMLIIGLNM 2.000 47 114 GSHLQRYLRI 2.000 48 298WPQTLGHLIN 2.000 49 406 LGLGLRHKAY 2.000 50 582 ITSAPGSRDY 2.000

Protein Motifs

Membrane associated proteinCalculated MW 78.6 kDa, pl 8.75

Multiple Transmembrane Domains

125P5C8 is modeled to have 10 transmembrane domains listed below

TABLE XIX Motif-bearing Subsequences of the 125P5C8 Protein N C Noterminal transmembrane region terminal 1 1 MTSLWREILLESLLGCVSWSLYH 23 242 LEGFSIAFLSPIFLTTTPFWKLV 64 3 94 MVLALGVSSSLIVQAVTWWSGSH 116 4 120YLRIWGFILGQIVLVVLRIWYTS 142 5 189 MASRPNWLLAGAAFGSLVFLTHW 211 6 238GGAVLLCLASGLMLPSCLWFRGT 260 7 269 TASAAGLLYLHTWAAAVSGCVFA 291 8 318LAMIFYLLEIFFCAWCTAFKFVP 340 9 350 DVLLGTMMLIIGLNMLFGP 368 10 390EKYMKLFLWLLVGVGLLGLGLR 411Protein Motifs present in 125P5C8:319-373(1051) Sodium: solute symporter family94-145(1009) Sodium: neurotransmitter symporter family122-137(1005) Sodium: dicarboxylate symporter family174-194(1009) Amiloride-sensitive sodium channel118-160(1014) Speract receptor (Scavenger receptor)

242-284(1086) Endothelin

N-glycosylation sites

Number of matches: 3

-   -   1 380-383 NSSK    -   2 455-458 NETG    -   3 536-539 NISG

Protein kinase C phosphorylation sites

Number of matches: 8

-   -   1 152-154 SNK    -   2 381-383 SSK    -   3 389-391 SEK    -   4 666-668 SEK    -   5 496-498 STR    -   6 538-540 SGK    -   7 389-391 SEK    -   8 666-668 SEK

Casein kinase II phosphorylation sites

Number of matches: 10

-   -   1 40-43 TGLE    -   2 170-173 TDGD    -   3 175-178 SKPE    -   4 445-448 SSLE    -   5 457-460 TGAD    -   6 463-466 TILE    -   7 606-609 TDHD    -   8 629-632 SHAE    -   9 634-637 SDSE    -   10 677-680 SYKE

Tyrosine kinase phosphorylation sites

Number of matches: 2

-   -   1 610-617 RWCEYIMY    -   2 644-652 RIPDDPTNY

N-myristoylation sites

Number of matches: 7

-   -   1 79-84 GSIASF    -   2 99-104 GVSSSL    -   3 199-204 GAAFGS    -   4 268-273 GTASAA    -   5 287-292 GCVFAI    -   6 309-314 GTNPGK    -   7 341-346 GGVYAR

TABLE XX Frequently Occurring Motifs av. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleicacid-binding protein functions as transcription factor, nuclear locationprobable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase,generate terminal)/b6/petB superoxide ig 19% Immunoglobulin domaindomains are one hundred amino acids long and include a conservedintradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandemrepeats of about 40 residues, each containing a Trp-Asp motif. Functionin signal transduction and protein interaction PDZ 23% PDZ domain mayfunction in targeting signaling molecules to sub-membranous sites LRR28% Leucine Rich Repeat short sequence motifs involved in protein-protein interactions pkinase 23% Protein kinase domain conservedcatalytic core common to both serine/threonine and tyrosine proteinkinases containing an ATP binding site and a catalytic site PH 16% PHdomain pleckstrin homology involved in intracellular signaling or asconstituents of the cytoskeleton EGF 34% EGF-like domain 30-40amino-acid long found in the extracellular domain of membrane-boundproteins or in secreted proteins rvt 49% Reverse transcriptase(RNA-dependent DNA polymerase) ank 25% Ank repeat Cytoplasmic protein,associates integral membrane proteins to the cytoskeleton oxidored_q132% NADH- membrane associated. Involved in protonUbiquinone/plastoquinone translocation across the membrane (complex I),various chains efhand 24% EF hand calcium-binding domain, consists ofa12 residue loop flanked on both sides by a 12 residue alpha-helicaldomain rvp 79% Retroviral aspartyl protease Aspartyl or acid proteases,centered on a catalytic aspartyl residue Collagen 42% Collagen triplehelix repeat extracellular structural proteins involved in (20 copies)formation of connective tissue. The sequence consists of the G-X-Y andthe polypeptide chains forms a triple helix. fn3 20% Fibronectin typeIII domain Located in the extracellular ligand-binding region ofreceptors and is about 200 amino acid residues long with two pairs ofcysteines involved in disulfide bonds 7tm_1 19% 7 transmembrane receptorseven hydrophobic transmembrane regions, (rhodopsin family) with theN-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

1. An antibody or fragment thereof that specifically binds to a125P5C8-related protein.
 2. The antibody or fragment thereof of claim 1,that binds to a portion of the 125P5C8-related protein, wherein theportion is selected from the group consisting of amino acid residues65-93, 143-188, 261-268, 341-349, and 412-699 of SEQ ID NO:2.
 3. Theantibody or fragment thereof of claim 1, that binds to a portion of the125P5C8-related protein, wherein the portion is selected from the groupconsisting of amino acid residues 24-41, 117-119, 212-237, 292-317, and369-389 of SEQ ID NO:
 2. 4. The antibody or fragment thereof of claim 1,which is monoclonal.
 5. The antibody or fragment thereof of claim 1,which is labeled with a detectable marker.
 6. The antibody or fragmentthereof of claim 1, which is an Fab, F(ab′)2, Fv or Sfv fragment.
 7. Theantibody or fragment thereof of claim 1, which is a human antibody. 8.The antibody or fragment thereof of claim 1, which is conjugated with acytotoxic agent.
 9. The antibody or fragment thereof of claim 8, whereinthe cytotoxic agent is selected from the group consisting of radioactiveisotopes, chemotherapeutic agents and toxins.
 10. The antibody orfragment thereof of claim 9, wherein the radioactive isotope is selectedfrom the group consisting of ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re,¹⁵³Sm, ²¹²Bi, ³²P and radioactive isotopes of Lu.
 11. The antibody orfragment thereof of claim 9, wherein the chemotherapeutic agent isselected from the group consisting of taxol, actinomycin, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicine, gelonin,and calicheamicin.
 12. The antibody or fragment thereof of claim 9,wherein the toxin is selected from the group consisting of diphtheriatoxin, enomycin, phenomycin, Pseudomonas exotoxin (PE) A, PE40, abrin,abrin A chain, mitogellin, modeccin A chain, and alpha-sarcin.
 13. Theantibody or fragment thereof of any one of claim 1, wherein the antibodyor fragment thereof further comprises a pharmaceutically acceptablecarrier.
 14. A hybridoma that produces an antibody of claim
 4. 15. Asingle chain monoclonal antibody that the variable domains of the heavyand light chains of a monoclonal antibody of claim
 4. 16. A vectorcomprising a polynucleotide that encodes the single chain monoclonalantibody of claim 9.